P R IMA R Y R E S E A R CH A R T I C L E

Interactions among plants bacteria and fungi reduceextracellular enzyme activities under long-term N fertilization

Joseph E Carrara1 | Christopher A Walter12 | Jennifer S Hawkins1 | William

T Peterjohn1 | Colin Averill3 | Edward R Brzostek1

1Department of Biology West Virginia

University Morgantown WV USA

2Department of Ecology Evolution and

Behavior University of Minnesota St Paul

MN USA

3Department of Biology Boston University

Boston MA USA

Correspondence

Joseph E Carrara Department of Biology

West Virginia University Morgantown WV

USA

Email jocarraramixwvuedu

Funding information

Division of Graduate Education Grant

Award Number DGE-1102689 Division of

Environmental Biology GrantAward

Number DEB-0417678 DEB-1019522

National Science Foundation Graduate

Research Fellowship Long-Term Research in

Environmental Biology (LTREB) program at

the National Science Foundation NOAA

Climate and Global Change Postdoctoral

Fellowship Program administered by

Cooperative Programs for the Advancement

of Earth System Science (CPAESS)

University Corporation for Atmospheric

Research (UCAR)

Abstract

Atmospheric nitrogen (N) deposition has enhanced soil carbon (C) stocks in temper-

ate forests Most research has posited that these soil C gains are driven primarily by

shifts in fungal community composition with elevated N leading to declines in lignin

degrading Basidiomycetes Recent research however suggests that plants and soil

microbes are dynamically intertwined whereby plants send C subsidies to rhizo-

sphere microbes to enhance enzyme production and the mobilization of N Thus

under elevated N trees may reduce belowground C allocation leading to cascading

impacts on the ability of microbes to degrade soil organic matter through a shift in

microbial species andor a change in plantndashmicrobe interactions The objective of

this study was to determine the extent to which couplings among plant fungal and

bacterial responses to N fertilization alter the activity of enzymes that are the pri-

mary agents of soil decomposition We measured fungal and bacterial community

composition rootndashmicrobial interactions and extracellular enzyme activity in the rhi-

zosphere bulk and organic horizon of soils sampled from a long-term (gt25 years)

whole-watershed N fertilization experiment at the Fernow Experimental Forest in

West Virginia USA We observed significant declines in plant C investment to fine

root biomass (247) root morphology and arbuscular mycorrhizal (AM) coloniza-

tion (559) Moreover we found that declines in extracellular enzyme activity were

significantly correlated with a shift in bacterial community composition but not fun-

gal community composition This bacterial community shift was also correlated with

reduced AM fungal colonization indicating that declines in plant investment below-

ground drive the response of bacterial community structure and function to N fertil-

ization Collectively we find that enzyme activity responses to N fertilization are

not solely driven by fungi but instead reflect a whole ecosystem response whereby

declines in the strength of belowground C investment to gain N cascade through

the soil environment

K E YWORD S

arbuscular mycorrhizal fungi belowground carbon allocation extracellular enzymes microbial

community nitrogen fertilization plantndashmicrobial interactions

Received 22 June 2017 | Revised 9 November 2017 | Accepted 9 January 2018

DOI 101111gcb14081

Glob Change Biol 20181ndash14 wileyonlinelibrarycomjournalgcb copy 2018 John Wiley amp Sons Ltd | 1

1 | INTRODUCTION

Atmospheric nitrogen (N) deposition has enhanced the carbon (C)

sink in both the soils and vegetation of temperate forests (Frey

et al 2014 Janssens et al 2010 Thomas Canham Weathers amp

Goodale 2010) Aboveground responses to N are relatively straight-

forward with additional N inputs relieving N limitation to tree

growth (Thomas et al 2010) Belowground a synthesis of data

across a suite of N addition experiments showed that soil C stocks

in forests are typically enhanced by N fertilization due to declines in

soil respiration (Janssens et al 2010) While this response appears

robust across multiple forest types the proximate mechanism driving

these declines in soil respiration remains unclear Given that the

worldrsquos soils contain more C than the atmosphere and vegetation

combined understanding the key mechanisms driving the response

of forest soil C to shifts in N input regimes is critical to predicting

future rates of C sequestration (Reay Dentener Smith Grace amp

Feely 2008)

Classically shifts in fungal community composition have been

viewed as the main driver of decomposition responses to enhanced

N inputs (Frey Knorr Parrent amp Simpson 2004 Morrison et al

2016 Wallenstein McNulty Fernandez Boggs amp Schlesinger

2006) According to this fungal-driven view elevated N inputs

reduce the abundance of white-rot fungi (phylum Basidiomycota)

one of the primary fungal guilds including both saprotrophs and

ectomycorrhizal species that can produce oxidative enzymes cap-

able of degrading lignin (Dix amp Webster 1995) The inability of

Basidiomycota to compete when N limitation is removed then leads

to declines in decomposition and increased soil organic matter

accumulation (Carreiro Sinsabaugh Repert amp Parkhurst 2000 Fog

1988 Waldrop Zak Sinsabaugh Gallo amp Lauber 2004 Zak

Holmes Burton Pregitzer amp Talhelm 2008) In support many stud-

ies report reduced ligninolytic enzyme activity in response to N fer-

tilization (DeForest Zak Pregitzer amp Burton 2004 Fog 1988 Frey

et al 2014 Saiya-Cork Sinsabaugh amp Zak 2002 Zak et al 2008)

However these enzymatic declines are not always coupled with a

corresponding decline in Basidomycota abundance (DeForest et al

2004 Hassett Zak Blackwood amp Pregitzer 2009) This apparent

disconnect between enzyme activity and fungal community compo-

sition suggests that soil C responses to N fertilization may not

solely be driven by elevated N directly inhibiting fungal growth and

activity

Emerging research suggests that enzyme activity declines in

response to N fertilization may reflect an integrated ecosystem

response that is driven by interactions among fungi bacteria and

plants Temperate forest trees allocate nearly 20 of net primary

production belowground to mycorrhizae and free-living microbes

in the rhizosphere which can influence microbial community com-

position stimulate extracellular enzyme production and enhance

plant access to N (Brzostek Fisher amp Phillips 2014 Hobbie

2006 Paterson Gebbing Abel Sim amp Telfer 2007 Yin et al

2013) When forest ecosystems are fertilized with N trees likely

reduce the C subsidies they send to rhizosphere microbes which

can alter the structure and function of not only fungal communi-

ties but also bacterial communities as well These potential shifts

in bacterial community composition are consequential given recent

evidence that bacteria can synthesize enzymes to degrade simple

and complex C (ie polyphenols lignin) in soil organic matter

(SOM Datta et al 2017 De Gonzalo Colpa Habib amp Fraaije

2016 Jackson Couger Prabhakaran Ramachandriya amp Canaan

2017) and that shifts in bacterial function can reduce enzyme

activities under N fertilization (Freedman Upchurch Zak amp Cline

2016) As such enzyme activity declines in response to N fertiliza-

tion may result from parallel shifts in plant bacterial and fungal

function whereby reductions in plant C allocation to rhizosphere

microbes indirectly alter fungal and bacterial community composi-

tion decrease the energy available to synthesize enzymes and

ultimately reduce the fungal and bacterial communityrsquos ability to

degrade SOM

While fungi have been shown to be important drivers of SOM

increases with N fertilization (DeForest et al 2004 Fog 1988 Frey

et al 2014 Saiya-Cork et al 2002 Zak et al 2008) focusing solely

on fungal responses may mask important interactions between other

components of the ecosystem that can alter enzyme activity

responses Thus the objective of this study was to examine the

extent to which couplings among plant fungal and bacterial

responses to N fertilization change extracellular enzyme activity (the

proximate agent of SOM decomposition Schimel amp Weintraub

2003) in a long-term (gt25 year) watershed-scale N fertilization

experiment in West Virginia United States To meet this objective

we linked assays of soil enzyme activities with measurements of fun-

gal and bacterial community composition and belowground C alloca-

tion across replicate plots in a reference and fertilized watershed

and used these measurements to parameterize a simple microbial

enzyme decomposition model to examine long-term effects of N fer-

tilization on soil C stocks

2 | MATERIALS AND METHODS

21 | Site description

The Fernow Experimental Forest (herein Fernow) is located in the

central Appalachian Mountains near the town of Parsons West Vir-

ginia (3903degN 7967degW) Mean annual precipitation is ~1460 mm

and mean annual temperature is 89degC (Kochenderfer 2006) Ambi-

ent wet N deposition at the site has been declining over time from

104 kg N ha1 year1 in 1989 (with an additional

4ndash5 kg N ha1 year1 dry deposition) to 50 kg N ha1 year1 in

2014 (Clean Air Status and Trends Network (Gilliam Yurish amp

Adams 1996) Soils are derived from sandstone and shale bedrock

and are classified as coarse textured Inceptisols (loamy-skeletal

mixed mesic Typic Dystrochrept) of the Berks and Calvin Series (Gil-

liam Turrill Aulick Evans amp Adams 1994)

To examine the impacts of N deposition on plantndashmicrobial

interactions we sampled soils and roots from a reference water-

shed receiving only ambient N deposition inputs and an N-

2 | CARRARA ET AL

fertilized watershed at the Fernow Both watersheds have stands

of similar age (~45 years old) and tree species assemblages Trees

gt25 cm in diameter at breast height (DBH) were cut in the

N-fertilized watershed (Watershed 3) in 1970 and recovered natu-

rally until 1989 when N treatments began (Peterjohn Adams amp

Gilliam 2017) N is added by aerial fertilization via helicopter at a

rate of 35 kg N ha1 year1 in the form of solid pellet (NH4)2

SO4 (Adams Edwards Wood amp Kochenderfer 1993) The

N-fertilized watershed is 343 ha with a south-facing aspect and

elevation ranging from 735 to 860 m (Gilliam et al 2016) The

reference watershed (Watershed 7) was clear cut from 1963 to

1967 (the upper half in 19631964 and the lower half in

19661977) and kept barren with herbicides until 1969 when it

began natural recovery (Kochenderfer 2006) This watershed is

240 ha with an east-facing aspect and elevation ranging from

731ndash850 m (Gilliam et al 2016) The watersheds are dominated

by arbuscular mycorrhizal associated (AM) tree species that include

tulip poplar (Liriodendron tulipifera) red maple (Acer rubrum) black

cherry (Prunus serotina) and sugar maple (Acer saccharum) which

together account for 59 of the basal area in the reference

watershed and 72 in the N-fertilized watershed Less abundant

ectomycorrhizal associated (ECM) tree species include sweet birch

(Betula lenta) red oak (Quercus rubra) and American beech (Fagus

grandifolia) which together account for 25 and 16 of the total

basal area in the reference and N-fertilized watershed

respectively

22 | Soil sampling protocol

We sampled soils on three dates in June July and August of 2015

in order to capture early peak and late growing season dynamics In

each watershed we sampled soils from an existing network of 10

randomly placed 25 9 25 m plots that have similar tree species

composition (Gilliam et al 1996) Three 10 9 10 cm organic horizon

(OH) samples were collected from each plot One OH sample was

used for root metrics and the other two for measures of soil biogeo-

chemistry After OH removal three 5-cm diameter soil cores were

extracted from each plot to a depth of 15 cm All samples were

stored on ice and returned to West Virginia University for further

processing

Within 24 hr of sampling mineral soil samples were separated

into bulk and rhizosphere fractions Rhizosphere soil was opera-

tionally defined as soil that adhered to roots upon removal from the

soil matrix and was carefully subsampled using forceps and homoge-

nized (sensu Phillips amp Fahey 2005) The remaining soil was consid-

ered mineral bulk soil After root and rhizosphere separation the

bulk and OH soil was homogenized by sieving through a 2-mm

mesh A subsample of each soil sample and fraction was stored at

80degC for assays of enzyme activity All fine roots (lt2 mm) were

washed in deionized water and subsequently dried and weighed to

determine standing root biomass Subsamples of roots from each

plot from the final sampling date were used to examine AM colo-

nization and root morphology

23 | Arbuscular Mycorrhizal colonization and rootmorphology

To determine the extent to which N fertilization altered AM colo-

nization we analyzed a subsample comprising ~13 of all roots from

the mineral and OH horizons for AM colonization Roots were

cleared by incubating in 10 potassium hydroxide for 48ndash96 hr at

room temperature depending on the pigmentation of the roots

Excess pigment was leached from roots by incubating in 85 etha-

nol for 24ndash48 hr This step was necessary due to the dark pigments

present in the woody tissue of our samples Any remaining pigment

was removed by soaking roots in ammoniahydrogen peroxide solu-

tion for 15 min Roots were prepared for staining by acidifying them

in 5 hydrochloric acid and then stained for 5 min in 005 trypan

blue (Comas Callahan amp Midford 2014)

We calculated AM colonization for each sample using the grid-

intersect method (Giovannetti amp Mosse 1980 McGonigle Miller

Evans Fairchild amp Swan 1990) Each sample was suspended in

water on a 1 9 1 cm gridded Petri dish and examined under a

stereoscopic microscope At each root-gridline intersect colonization

was determined by the presence of arbuscules or hyphae Percent

AM colonization was then calculated as the percent of total inter-

sects examined that had visible AM structures Given that roots with

high levels of AM colonization are likely to be more involved in

releasing C to rhizosphere microbes (Kaiser et al 2015) we used

the level of colonization by AM fungi as a metric of the degree to

which roots and microbes were actively coupled

We created digital images of root morphology using an LA2400

desktop scanner (WinRhizo Regent Instruments Inc) and then used

WinRhizo software to determine root length surface area average

diameter and number of forks of each sample All measurements

were expressed per g of root mass and scaled to a per m2 basis

based on total standing root biomass (gm2) at the plot level

(Table 1)

24 | Extracellular enzyme activity

For all soil fractions at each sampling date we assayed the potential

activity of hydrolytic enzymes that release nitrogen (N-Acetylgluco-

saminidase NAG) phosphorus (acid phosphatase AP) and simple

carbon (b-glucosidase BG) Given extracellular enzyme activity is the

rate-limiting step of the decomposition of organic matter we con-

sider these measurements a metric of decomposition (Schimel amp

Weintraub 2003) Soil extracellular enzyme activity has been linked

to mass-loss rates of wood (Sinsabaugh et al 1992 1993) and soil

C-mineralization (Brzostek Greco Drake amp Finzi 2013 Hernandez

amp Hobbie 2010) After thawing 1 g of each sample was homoge-

nized in 50 mM sodium acetate buffer (pH 50) Hydrolytic enzyme

activities were determined using a fluorometric microplate assay

with methylumbelliferone-linked substrates In addition we assayed

for activities of liginolytic oxidative enzymes phenol oxidase and

peroxidase using a colorimetric microplate assay based on the

hydrolysis of L-34-dihydroxyphenylalanine linked substrates (Saiya-

CARRARA ET AL | 3

Cork et al 2002) These assays were performed under saturated

substrate conditions wherein the concentration of enzyme limits

activity

We measured ambient proteolytic activity of each soil sample

following a protocol adapted from Brzostek and Finzi (2011) Each

subsample (2ndash3 g) received 10 ml of 005 M sodium acetate buffer

(pH 50) and 400 ll toluene to inhibit microbial reuptake of amino

acids Initial samples were immediately terminated by adding 3 ml of

trichloroacetic acid Incubated samples were shaken at room temper-

ature for 4 hr prior to termination Proteolysis was expressed as the

difference between incubated and initial amino acid concentrations

Amino acid concentrations were measured following the fluorometric

OPAME method (Jones Owen amp Farrar 2002) where fluorescence

of each sample was a function of amino acid concentration calcu-

lated from a glycine standard curve In contrast to the other enzyme

assays described above this proteolytic enzyme assay is run at ambi-

ent substrate conditions and reflects the limitation of activity by

both enzyme and substrate concentrations

25 | Bacterial and fungal community composition

Soil microbial DNA was extracted from samples collected in July 2015

using the Zymo soil microbe mini prep kit (Zymo Research Corpora-

tion Irvine CA USA) and quantified on a Nanodrop 3300 (Thermo

Scientific Wilmington DE USA) Due to logistical constraints we

chose the July sampling date in order to examine microbial community

composition at the peak of the growing season The fungal ITS1 and

bacterial 16S loci were chosen for amplification via polymerase chain

reaction (PCR) in order to delineate microbial community composition

and the corresponding shifts associated with N amendment All PCR

reactions contained 1ndash5 ng template DNA 19 Mg-included Taq

buffer 08 mM dNTPs 005 U of Taq polymerase (Denville Scientific

South Plainfield NJ USA) and 04 pM each of either ITS1-F_KYO1

(Toju Tanabe Yamamoto amp Sato 2012) and ITS2 primers (Gardes amp

Bruns 1993 White Bruns Lee amp Taylor 1990) for fungal amplifica-

tion or S-D-Bact-0341-b-D-17 and S-D-Bact-0785-a-A-21 (Klind-

worth et al 2013) for bacterial analysis Primers were modified to

include 197 unique 8 bp index sequences (for individual sample ldquobar-

codingrdquo) in addition to Illumina adapters and Nextera sequencing

motifs (sequences available upon request) Reaction conditions were

as follows 95degC denaturation for 3 min 30 cycles of 98degC for 20 s

61degC for 30 s 72degC for 30 s followed by a final extension at 72degC for

5 min All amplification products were resolved on a 1 agarose gel

extracted with a sterile razor and purified using the PureLink Quick

Gel Extraction Kit (Invitrogen Carlsbad CA USA) Purified products

were quantified via Qubit pooled in equimolar amounts and

sequenced in concert on the Illumina MiSeq at the West Virginia

University Genomics Core Facility (Morgantown WV)

Paired end sequences were merged and barcodes and primers

trimmed using the Quantitative insights into Microbial Ecology

(QIIM) pipeline version 190 (Caporaso et al 2010) Using a Phred

score cut-off of 30 reads with less than 75 high quality base calls

were excluded from further analysis We also discarded sequences

with three or more adjacent low-quality base calls Sequences were

clustered into operational taxonomic units (OTUs) using the

USEARCH algorithm with an open reference OTU picking strategy

using a 97 sequence similarity threshold (Edgar 2010) Bacterial

16S sequences were clustered against the Greengenes reference

database (DeSantis et al 2006) and fungal sequences were clus-

tered against the UNITE ITS database (K~oljalg et al 2013) Taxon-

omy was assigned using the RDP 9 22 classifier (Wang Garrity

Tiedje amp Cole 2007) and associated Greengenes and UNITE

Fine root metric Control +N Change

Organic horizon

Root biomass (gmsup2) 3083 407 5213 850 691

Mycorrhizal colonization () 728 36 609 001 163

Specific root length (cmg) 203869 26099 199299 17960 224

Surface area (cmsup2g) 5188 532 8563 2822 251

Average diameter (mm) 044 002 043 002 42

Root volume (cmsup3g) 185 010 185 017 024

Forksg 6027 739 5967 910 098

Mineral soil

Root biomass (gmsup2) 31923 2631 24051 4820 247

Mycorrhizal colonization () 753 58 332 67 559

Specific root length (cmg) 124057 9598 97658 9393 213

Surface area (cmsup2g) 16713 881 12525 847 251

Average diameter (mm) 044 002 043 002 65

Root volume (cmsup3g) 184 012 130 057 294

Forksg 2598 351 2091 183 195

Mean values of all sampling dates are accompanied by standard error

Significant differences between treatments (p lt 05)

TABLE 1 Fine root and mycorrhizalmetrics in bulk and organic horizon soilsunder ambient and long-term N-fertilizedconditions

4 | CARRARA ET AL

reference databases To determine the effect of N fertilization on

overall species composition the top four abundant fungal phyla

(Ascomycota Basidiomycota Chytridiomycota and Zygotmycota as

well as lsquounclassifiedrsquo) were examined for shifts in relative abundance

To further analyze fungal and bacterial community composition

each OTU table was proportionally normalized such that the per OTU

sequence count within a sample was divided by the total number of

sequences detected in a given sample generating a proportion on the

interval [01] This proportional normalization has been demonstrated

to be sufficiently accurate when using the BrayndashCurtis similarity met-

ric (Weiss et al 2017) We then used normalized OTU tables to calcu-

late BrayndashCurtis similarity using the vegdist function within the vegan

package for R statistical software (Oksanen et al 2015 R Core Team

2017) Community similarity was analyzed as a function of watershed

soil horizon and their interaction using the adonis function within the

vegan package If interactions were not significant then they were

removed and we only analyzed main effects

To understand how microbial community composition related to

variation in soil enzyme profiles we created similarity matrices of

soil enzymes We calculated BrayndashCurtis similarity of enzyme profiles

using all enzymes using the vegdist function in vegan We then per-

formed nonmetric multidimensional scaling (NMDS) to generate

NMDS scores for both microbial and enzymatic profiles We tested

for relationships between microbial communities and enzymatic pro-

files in bulk and rhizosphere soil separately by comparing the first

NMDS axes of microbial and enzymatic profiles using linear regres-

sion in SAS JMP vs 1002 (SAS Institute Cary NC USA) We fur-

ther examined the relationship between microbial communities and

enzymatic profiles across all soil horizons by comparing the first

NMDS axes of microbial and enzymatic profiles using linear regres-

sion across both watersheds

To assess the relative importance of belowground C allocation

(ie AM colonization) on fungal and bacterial community composi-

tion we explored whether there were correlations between AM

colonization and the first NMDS axis of bacterial and fungal commu-

nity composition in bulk and rhizosphere soil separately using SAS

JMP vs 1002 (SAS Institute Cary NC USA)

26 | Net mineralization and nitrification

For all soil fractions at each sampling date we measured the rate of

net N mineralization and net nitrification within 48 hours of collec-

tion Net N mineralization and nitrification rates were expressed as

the difference in the 1 M KCl extractable pool sizes of NHthorn4 and

NO3 between an initial sample and a sample that was aerobically

incubated in the lab for 14 days at room temperature and under

constant soil moisture (Finzi Van Breemen amp Canham 1998) To

perform the extractions 50 ml of 1 M KCl was added to a jar con-

taining 5 g of each soil sample Samples were sealed and shaken for

30 min and allowed to settle for 2 hr Samples were filtered using

Pall Supor 450 filters via syringe into 20-ml vials and stored at

20degC prior to analysis by flow injection (Lachat QuikChem 8500

series 2)

27 | Statistical analysis

To determine the extent to which N fertilization altered enzyme

activities N mineralization nitrification and root metrics we per-

formed a three-way analysis of variance (ANOVA) in SAS JMP vs

1002 (SAS Institute Cary NC USA) and used date watershed and

soil horizon as factors Although we included date in our statistical

models we present growing season means in the results reflecting

our focus on treatment rather than seasonal effects Post hoc multi-

ple comparisons were also made among watersheds dates and soil

fractions using the TukeyndashKramer HSD test

28 | Modeling potential impacts of plantndashmicrobiallinkages on soil decomposition

To further examine the extent to which linkages between plants and

soil microbes drive soil enzyme responses to N fertilization as well

as to estimate potential impacts on soil C stocks we used a micro-

bial enzyme decomposition model (Allison Wallenstein amp Bradford

2010) that was modified in Cheng et al (2014) to include seasonal

differences in soil temperature the inputs of root and leaf litter and

root transfers of C to the rhizosphere For leaf and root litter inputs

we used the mean observed litterfall rate from 1998 to 2015 for

each watershed (Kochenderfer 2006 Peterjohn et al 2017) and our

own measurements of root biomass with the assumption that roots

in both watersheds turnover once each year (McCormack Adams

Smithwick amp Eissenstat 2017) For both roots and leaves we

assumed that turnover primarily occurred during the leaf senescence

in the fall It is important to note there are no significant differences

in leaf litterfall rates between the two watersheds (WS3 = 143 g C

m2 WS7 = 146 g Cm2 Kochenderfer 2006) Finally we used pub-

lished values of AM root exudation rates on a per g root basis (Yin

Wheeler amp Phillips 2014) to estimate root C transfers assuming

that these inputs occurred only during the growing season Using

these model inputs we then ran two scenarios (1) the fertilized and

control watersheds had similar root C exudation rates per g of root

and (2) N fertilization reduced exudations rates by 25 We chose

this reduction in exudation rates to be conservative as it represents

around half of the decline in AM colonization we observed in the

fertilized watershed All model simulations were run for 30 year to

achieve steady state

3 | RESULTS

31 | Fine root metrics

Fine root biomass and the extent of mycorrhizal symbiosis in the

bulk mineral soil layer were significantly lower in the N-fertilized

watershed (Table 1) Overall the bulk mineral soil in the N-fertilized

watershed had 247 less standing fine root biomass than the refer-

ence watershed This reduction in fine root biomass was accompa-

nied by decreases in fine root surface area volume and the amount

of root forks per g Additionally N fertilization reduced AM

CARRARA ET AL | 5

colonization of fine roots by 559 Given the inherent limitations of

measuring root production using minirhizotron tubes or ingrowth

cores we use fine root biomass and AM colonization as proxies for

plant C allocation to gain nutrients While root turnover may be

enhanced under N fertilization we base the assumption that fine

root biomass is synonymous with belowground C allocation to fine

roots on our observations that fine root biomass was consistently

lower in the fertilized than control watersheds over all three of the

sequentially cored soil sampling dates during the growing season In

addition AM fungi have been shown to act as conduits for photo-

synthate C to rhizosphere microbes and as such represent an impor-

tant belowground flux of C (Kaiser et al 2015) Thus reduced

investment in AM colonization may also lead to reduced transfers of

photosynthate C to the rhizosphere resulting in an overall decline in

belowground C allocation

By contrast in the OH standing fine root biomass was larger in

the N-fertilized watershed although root biomass in the OH horizon

is markedly lower than in the bulk mineral soil (Table 1) However

like the bulk mineral soil AM colonization of OH roots was signifi-

cantly higher in the reference watershed (728 colonized) than the

N-fertilized watershed (609 colonized)

32 | Extracellular enzyme activity

Nearly all hydrolytic enzyme activities were lower under elevated N

conditions across all three soil fractions BG activity was significantly

lower in the N-fertilized watershed in both the bulk mineral (52

decrease) and OH (17 decrease) soil fractions (p lt 05 Figure 1a)

N fertilization significantly lowered AP activity in the bulk (12) and

rhizosphere (36) fractions of the mineral soil (p lt 05 Figure 1b)

Similarly N amendment significantly lowered NAG activity in both

bulk and rhizosphere fractions of the mineral soil by 41 and 37

respectively (p lt 05 Figure 1c) However BG activity in the rhizo-

sphere of the mineral soil was not significantly different between

0

04

08

12

16

Bulk Rhizo

AP

(m

olmiddot[

gdr

yso

il]ndash1

middot hr

ndash1)

0

1

2

3

4

5

OH0

01

02

03

04

Bulk Rhizo

BG

(m

olmiddot[g

dry

soi

l]ndash1

middot hr

ndash1)

0

05

1

15

2

OH

0

003

006

009

012

Bulk Rhizo

NA

G (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)

0

02

04

06

08

1

OH

0

04

08

12

16

Bulk Rhizo0

10

20

30

40

50

OH

0

025

05

075

1

Bulk Rhizo0

1

2

3

OH

0

1

2

3

4

5

Bulk Rhizo

Pero

xida

se (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)

0

1

2

3

4

OH

(a) (b)

(c) (d)

(e) (f)

Phen

ol o

xida

se (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)Pr

oteo

lysi

s (

gA

A-N

middot[g

dry

soil]

ndash1 middot

hrndash1

)

Reference

+N

F IGURE 1 N fertilization reduceshydrolytic and oxidative enzyme activitiesValues are the overall seasonal meanenzyme activities (mean SE) of (a) BG(b) AP (c) NAG (d) phenol oxidase (e)peroxidase and (f) proteolysis for each soilfraction (ie bulk rhizosphere and organichorizon) measured in June July andAugust 2015 across all plots (n = 10 plotsper watershed) Asterisks indicatesignificant differences in enzyme activitybetween watersheds within soil fractions(p lt 05) Note difference in scale betweenOH vs rhizosphere and bulk soil fractionsAA-N is amino acid nitrogen

6 | CARRARA ET AL

watersheds In addition AP and NAG activities in the OH horizon

did not vary by treatment

Liginolytic oxidative enzyme activities were also generally lower

in the N-fertilized watershed Phenol oxidase activity was signifi-

cantly lower in bulk (45 decrease) rhizosphere (49 decrease) and

OH horizons (57 decrease) of the fertilized watershed relative to

the reference watershed (p lt 05 Figure 1d) Peroxidase activity was

lower in the fertilized watershed in the bulk rhizosphere and OH

soil fractions with reductions of 30 25 and 36 respectively

(p lt 05 Figure 1e) Proteolytic enzyme activity was consistently

lower in each soil fraction under N fertilization with a 48 decrease

in bulk 56 decrease in rhizosphere and 40 decrease in OH

proteolysis (Figure 1f)

33 | Microbial community composition

When fungal taxonomic units were aggregated to the phylum level

there were no significant changes in the relative abundance of the

four most common fungal phyla or the unclassified group in any soil

horizon (Figure S1) When bacterial taxonomic units were aggregated

to the phylum level there were limited shifts in the relative abun-

dance of the seven most common bacterial phyla Relative abun-

dance of Actinobacteria was higher in the OH soil of the fertilized

watershed (Figure S2a) Relative abundance of Proteobacteria was

lower in the fertilized bulk soil and relative abundance of Firmicutes

was higher in both rhizosphere and bulk soil in the fertilized water-

shed (Figure S2bc)

Adonis analysis of bacterial communities revealed significant

effects for watershed soil horizon (p lt 001) and their interaction

(p = 02 total model R2 = 32) Within watersheds post hoc compar-

isons showed OH communities were different than bulk and rhizo-

sphere communities (Figure S3ab) There was no difference

between bulk and rhizosphere communities (Figure S3ab) Across

watersheds bacterial community composition differed in all soil frac-

tions such that OH rhizosphere and bulk soil exhibited unique com-

munities in the N-fertilized watershed compared to the reference

(Figure 2ace)

Adonis analysis of fungal communities revealed significant effects

of watershed and soil horizon (p lt 001 total model R2 = 12) but

not their interaction Within watersheds post hoc comparisons of

fungal communities showed OH communities were different than

bulk and rhizosphere communities in both watersheds but there was

no difference between bulk and rhizosphere communities

(Figure S3cd) Across watersheds fungal community composition

within OH and bulk soil fractions was significantly different (p lt 01

Figure 2bd) However there was no significant difference in fungal

communities between watersheds in rhizosphere soil (Figure 2f)

Comparison of bacterial and enzymatic NMDS scores across all

soil horizons showed a positive relationship between bacterial com-

munities and enzyme profiles (R2 = 48 p lt 01 Figure S4a) Compar-

ison of fungal and enzymatic NMDS scores across all soil horizons and

both watersheds showed no significant relationship between fungal

communities and enzyme profiles (Figure S4b) Across watersheds

comparison of bacterial and enzymatic NMDS scores showed a nega-

tive relationship between bacterial communities and enzyme profiles

in the bulk soil (R2 = 36 p lt 05 Figure 3a) but no significant rela-

tionship in rhizosphere soil Comparison of fungal and enzymatic

NMDS scores across watersheds showed no significant relationships

in either bulk or rhizosphere soil (Figure 3b) Linear regression of

AM colonization and the first bacterial NMDS axis resulted in a posi-

tive linear relationship in bulk soil (R2 = 063 p lt 001 Figure 3c) but

not rhizosphere soil There were no significant linear relationships

between AM colonization and the first fungal NMDS axis in either

soil fraction (Figure 3d)

34 | Net N mineralization and nitrification

The only significant difference we found in rates of nitrogen cycling

was that N mineralization and net nitrification in the OH were 40

and 51 higher in the fertilized watershed than the reference N

transformation rates did not significantly differ between watersheds

in either the bulk or rhizosphere soil fractions (Figure 4ab)

35 | Modeling potential impacts of plantndashmicrobiallinkages on soil decomposition

When we ran the model to steady state we found that reductions

in root biomass and root C transfers to the rhizosphere in the N-fer-

tilized watershed reduced microbial enzyme pools by ~16 and

enhanced soil C by ~3 compared to the reference watershed

(Table 2) Even though exudation rates on a per g root basis were

the same the fertilized watershed had lower overall exudations rates

at the ecosystem scale than the control watershed because of its

lower root biomass (Table 2) When we reduced exudation rates by

25 on a per g root basis in the fertilized watershed there was a

further exacerbation in the reduction in microbial enzyme activity to

a ~28 decline and a larger increase in soil C by ~20 (Table 2)

4 | DISCUSSION

Here we provide evidence that coupled interactions among plants

fungi and bacteria play an important role in enzyme activity

responses to N fertilization For fungi we observed distinct shifts in

fungal community structure in response to N fertilization (Figure 2b

d) but we found no evidence for a link between these shifts and

extracellular enzyme activity (Figure 3b) By contrast we found that

bacterial community composition shifts under N fertilization are cor-

related with declines in enzyme activity (Figure 3a) and that these

compositional shifts are tightly coupled to reductions in plant C allo-

cation to roots and mycorrhizal fungi (Figure 3c) Overall these

results suggest that N fertilization drives an integrated ecosystem

response whereby reductions in plant C allocation to roots and AM

fungi feedback on bacterial community structure and function

While whole-watershed fertilization at the Fernow results in a

pseudoreplicated experimental design (Hurlbert 1984) we conclude

CARRARA ET AL | 7

that the effects we measured are driven by N fertilization rather

than pre-existing differences between these adjacent watersheds

for four main reasons First soil chemistry (ie soil pH cation

exchange capacity nutrient content etc) were similar at the begin-

ning of the experiment (Adams amp Angradi 1996) Second the

amount of N added to the watershed yearly was originally chosen in

1989 to approximately double ambient N deposition rates but is

now more than quadruple current rates and as such it seems unli-

kely that this does not incur a biogeochemical response Third the

results from this watershed study are consistent with measurements

we made during the same year in a replicated N fertilization study

lt2 km away from these watersheds (the Fork Mountain Long-Term

Soil Productivity Plots Adams Burger Zelazny amp Baumgras 2004)

In this small-scale replicated study N fertilization reduced fine root

biomass and ligninolytic enzyme activity (Figure S5) Finally this

work builds upon other research at the Fernow that has found the

N-fertilized watershed had lower rates of litter decomposition

(Adams amp Angradi 1996) reduced understory richness (Gilliam et al

1994 2016 Walter Adams Gilliam amp Peterjohn 2017) and altered

N cycling (Adams et al 1993 Burnham Cumming Adams amp Peter-

john 2017 Gilliam Yurish amp Adams 2001 Gilliam et al 1996

2016) compared to the reference watershed

While we did observe significant declines in enzyme activity

across all three soil fractions particularly for the liginolytic enzymes

(Figure 1andashf) these declines were not correlated with significant

shifts in fungal community composition (Figure 3b) The lack of a

clear link between enzyme declines and changes in the fungal com-

munity does not support the prevailing paradigm that white-rot

Basidiomycota are the dominant cause of a decline in enzyme activi-

ties following N additions (Edwards Zak Kellner Eisenlord amp Pregit-

zer 2011 Fog 1988 Freedman Romanowicz Upchurch amp Zak

2015 Morrison et al 2016) It is possible that fungal enzyme activ-

ity response to N fertilization is independent of community composi-

tion (ie investment in enzyme activity declines with no change in

community structure) however microbial community composition

has been linked to catabolic functioning and enzyme activities across

N gradients and seasons (Fierer et al 2011 Vorıskova Brabcova

Cajthaml amp Baldrian 2014) Furthermore our data indicate that

ndash008

0

008

016

024

032

ndash09 ndash06 ndash03 0 03 06

Rhi

zo f

ungi

NM

DS2

Rhizo fungi NMDS1

ndash04

ndash02

0

02

04

ndash02 ndash01 0 01 02 03 04

Rhi

zo b

acte

ria

NM

DS2

Rhizo bacteria NMDS1

ndash02

ndash01

0

01

02

03

ndash02 0 02 04 06

Bul

k ba

cter

ia N

MD

S2

Bulk bacteria NMDS1

ndash04

ndash032

ndash024

ndash016

ndash008

0

ndash04 ndash02 0 02 04

OH

fun

gi N

MD

S2

OH fungi NMDS1

ndash06

ndash04

ndash02

0

02

ndash05 ndash04 ndash03 ndash02 ndash01 0

OH

bac

teri

a N

MD

S2

OH bacteria NMDS1

Reference

+N

ndash02

ndash01

0

01

02

03

04

ndash06 ndash04 ndash02 0 02 04

Bul

k fu

ngi N

MD

S2

Bulk fungi NMDS1

(a) (b)

(c) (d)

(e) (f)

plt01

plt01

plt01

plt01

plt01

F IGURE 2 N fertilization alteredbacterial community composition in OH (a)bulk (c) and rhizosphere (e) soils andfungal community composition in OH (b)and bulk (d) soils but not rhizosphere (f)All community data were obtained for eachsoil fraction in July 2015 (n = 10 plots perwatershed) p values indicate significantdifferences between the N-fertilized andreference community

8 | CARRARA ET AL

shifts in bacterial community composition under elevated N are cor-

related with reduced enzyme activity at the Fernow (Figure 3a)

While changes in bacterial community may be influenced by overall

fungal community composition these shifts in bacterial community

composition are tightly coupled to AM colonization (a metric of

belowground C allocation) suggesting that plant responses to N fer-

tilization feedback on bacterial community structure and function

(Figure 3c) While we cannot rule out that N-induced declines in

total fungal biomass led to reductions in liginolytic enzyme activity

(DeForest et al 2004 Frey et al 2004 Treseder 2008 Wallenstein

et al 2006) the dominance of AM trees in our plots whose inor-

ganic nutrient economy is largely driven by bacteria suggests that

free-living fungi may not be an important driver of N deposition

responses in AM-dominated systems (Cheeke et al 2017 Phillips

Brzostek amp Midgley 2013)

In contrast it appears that the declines in enzyme activity we

observed appear to be the result of a cascade of ecosystem

responses affecting both microbial community composition and plant

C allocation belowground The N-induced declines in fine root bio-

mass AM colonization and root morphology all indicate that there

was a reduction in the investment of C belowground by trees to gain

nutrients (Table 1) While previous research has shown that below-

ground C allocation is inversely correlated with N availability (Bae

Fahey Yanai amp Fisk 2015 Haynes amp Gower 1995 Phillips amp Fahey

2005) the tight linkage between the first bacterial NMDS axis and

AM colonization suggest that N-induced declines in belowground

C allocation caused a shift in the bacterial community composition

and subsequent reductions in enzyme activity (Figure 3ac) This

result also supports the emerging view that roots are active engi-

neers of microbial activity (Brzostek et al 2013 Finzi et al 2015

Yin et al 2014) and highlights the need to integrate root-microbial

interactions into our conceptual and predictive modeling frameworks

of how forest ecosystems respond to global change (Johnson

Gehring amp Jansa 2016 Terrer Vicca Hungate Phillips amp Prentice

2016)

An additional impact of the N fertilization treatment is that it

reduced soil pH (as measured in a 001 M calcium chloride buffer) of

the upper 5 cm of mineral soil in the fertilized watershed to 34

compared to 38 in the control watershed (Peterjohn unpublished

data) Soil pH is an important control on microbial community diver-

sity (Fierer et al 2012 Kaiser et al 2016 Lauber Hamady Knight

amp Fierer 2009) microbial biomass and enzyme activities (Rousk amp

Baath 2011 Sinsabaugh 2010) Thus the decline in soil pH may

account for a portion of the enzyme reductions we observed under

N fertilization To address this we assayed the sensitivity of phenol

oxidase and peroxidase enzyme activity in organic horizon bulk and

rhizosphere soils from the control watershed to three different levels

of pH that spanned a 1 unit shift (data not shown) Peroxidase activ-

ity was insensitive to pH (slope = 074 r2 = 05 p gt 05) While

phenol oxidase activity did significantly increase as pH increased

(slope = 010 r2 = 053 p lt 05) this sensitivity would only account

for a 10 decline in activity Given that we observed a 50 decline

in phenol oxidase in the treatment watershed coupled with the

greater overall importance of peroxidase enzymes in our study (ie

nearly an order of magnitude higher activity) it appears that pH is

an important but secondary driver of the enzyme activity responses

we observed

Over longer time scales the reductions in root and microbial

activity we observed at the Fernow may have important implications

for soil C stocks In our model simulation we found that feedbacks

between reductions in root biomass and enzyme production have

the potential to drive nearly a 3 increase in soil C stocks (Table 2)

When these were coupled with a 25 reduction in specific root C

exudation rates soil C in the fertilized watershed increased by nearly

20 over the 30-yr simulation This model was designed to be theo-

retical and as such it is used here to show the sensitivity of the

y = 0004x ndash 007R = 63

ndash02

0

02

04

06

0 25 50 75 100Bul

k B

acte

ria

OT

U N

MD

S1

AM Colonization ()

y = ndash0159x + 013R = 360

004

008

012

016

02

ndash02 0 02 04 06

Enz

yme

NM

DS

1

Bacteria OTU NMDS1

+NReference

0

004

008

012

016

02

ndash06 ndash04 ndash02 0 02 04

Enz

yme

NM

DS

1

Fungal OTU NMDS1

ndash06

ndash04

ndash02

0

02

04

0 25 50 75 100

Bul

k Fu

ngi

OT

U N

MD

S1

AM Colonization ()

plt05

plt001

(a)

(c)

(b)

(d)F IGURE 3 Bacterial but not fungalcommunity composition is correlated withthe first NMDS axis of enzyme activity (aamp b) Percent AM colonization is correlatedwith bacterial community composition (c)but not fungal community composition (d)Data presented are bulk soil communitycomposition data from July 2015 (n = 10plots per watershed) p values indicatesignificance of correlation

CARRARA ET AL | 9

system to the perturbations in plant inputs and is not meant to be

quantitatively predictive However this simple modeling effort pro-

vides compelling evidence that N-induced shifts in rootndashmicrobial

interactions have the potential to alter not only microbial production

of soil enzymes but also the size of the soil C pool To generate the

necessary data for fully integrating root-microbial interactions into

more sophisticated ecosystem models future research should couple

direct measurements of belowground C allocation responses to N

fertilization (eg total belowground C allocation root exudation)

with the resulting impacts on microbial activity

Given the prevailing paradigm that free-living fungi drive soil

enzyme activity and subsequent decomposition responses to N fer-

tilization (Carreiro et al 2000 Fog 1988 Waldrop et al 2004 Zak

et al 2008) our results raise an important question of why shifts in

plant C allocation bacterial community structure and enzyme activi-

ties were tightly coupled at the Fernow The divergent results

between the Fernow and other long-term fertilization sites may be

the result of differences in ambient N deposition and tree species

composition The Fernow is in a region that has historically received

some of the highest levels of N deposition in the United States

(Driscoll et al 2001) which may have enhanced bacterial dominance

before treatment began in 1989 By contrast N fertilization experi-

ments that have linked reductions in soil C decomposition to fungal

community composition and activity tend to be located in areas with

lower historical N deposition rates (Edwards et al 2011 Freedman

et al 2015 Frey et al 2004 Morrison et al 2016 Wallenstein

et al 2006) Second the Fernow is predominantly dominated by

AM trees whereas most N fertilization experiments have tended to

occur in ecosystems dominated by ECM trees with ECM-dominated

sites comprising ~80 of the studies included in the Janssens et al

(2010) meta-analysis AM trees show greater growth enhancements

with N addition (Thomas et al 2010) are characterized by lower

fungal to bacterial ratios (Cheeke et al 2017) and are less depen-

dent on plantndashmicrobial interactions to access N than ECM trees

(Brzostek Dragoni Brown amp Phillips 2015) As such in AM-domi-

nated systems like the Fernow bacteria may outweigh fungi in con-

trolling soil decomposition responses to N fertilization Additionally

evidence that ECM trees rely more on roots and rhizosphere

microbes to access N than AM trees suggests that N fertilization

may lead to greater declines in belowground C allocation and

enzyme activity than observed at the Fernow (Brzostek et al 2015

Yin et al 2014) Moving forward the equal distribution of ECM and

AM trees across the temperate forest landscape highlights a critical

need to investigate ECM and AM stand responses to N fertilization

within the same ecosystem (Midgley amp Phillips 2014)

Although enzyme declines were observed across all three soil

fractions (Figure 1andashf) N cycling shifts were only observed in the

OH where rates were elevated by nearly 50 and 40 for mineral-

ization and nitrification respectively (Figure 4ab) OH and mineral

soils differ in key biogeochemical traits and this may contribute to

these divergent N cycling responses In temperate forests OH

0

05

1

15

Bulk Rhizo

Net

N m

iner

aliz

atio

n (

gN

middotgndash1

middotday

ndash1)

Control

+N

0

3

6

9

12

15

18

OH

0

05

1

15

Bulk Rhizo

Net

nitr

ific

atio

n ((

gN

middotgndash1

middotday

ndash1)

0

2

4

6

8

10

12

OH

(a)

(b)

Reference

F IGURE 4 N fertilization increases (a) N mineralization and (b)nitrification in the OH but not the mineral soil Values are theoverall watershed-level mean rates (mean SE) measured in JuneJuly and August 2015 for each soil fraction (n = 10 plots perwatershed) Asterisks indicate significant differences betweentreatments (p lt 05)

TABLE 2 At model steady state fertilization reduced microbialenzyme pools by ~16 and enhanced soil C by ~3

Watershed ExudationEnzyme pool(mgcm3)

Soil C pool(mgcm3)

Control No change 00136 18473

Fertilized No change 00114 19092

25 reduction 00097 22116

When root C exudation was reduced 25 on a per g root basis in the

fertilized watershed there was a further exacerbation in the reduction in

microbial enzyme activity to a ~28 decline and a larger increase in soil

C by ~20

10 | CARRARA ET AL

typically have rapid N cycling rates reflecting greater organic matter

content and root densities than mineral soils (Brzostek amp Finzi

2011) Shifts in the ratio of gross immobilization to gross mineraliza-

tion may also play a role Net N mineralization rates increased in the

OH despite N fertilization induced declines in proteolytic and chiti-

nolytic enzyme activity that produce N monomers for microbial

uptake While we do not have data on gross N cycling we hypothe-

size that declines in gross microbial N immobilization due to reduced

N demand may have outpaced declines in gross N mineralization in

the fertilized watershed Regardless of the exact mechanism it is

important to put the OH results into context on a mass per unit

area basis the OH is less important to total ecosystem N cycling

than the underlying mineral soil

Much of the research on soil N fertilization has focused on

aboveground responses such as litter input and quality or fungal

community shifts to explain widely observed reductions in enzyme

activities and subsequent soil decomposition (Edwards et al 2011

Frey et al 2004 Morrison et al 2016 Sinsabaugh et al 2008

Waldrop et al 2004 Wallenstein et al 2006) Our results provide

evidence that enzyme activity declines under long-term N fertiliza-

tion at the Fernow are driven by a cascade of ecosystem responses

whereby reductions in belowground plant C investment lead to

shifts in bacterial community structure and a decline in their ability

to degrade soil organic matter Thus there is a need to integrate

plantndashmicrobial interactions into our current conceptual and predic-

tive models of N deposition impacts on temperate forests in order

to forecast soil C responses to shifting N deposition regimes

ACKNOWLEDGEMENTS

We acknowledge Mary Beth Adams Tom Schuler and the US Forest

Service staff at the Fernow Experimental Forest for logistical assis-

tance and access to the experimental watersheds and the LTSP site

This work was also supported by the National Science Foundation

Graduate Research Fellowship to Joseph Carrara under Grant No

DGE-1102689 and by the Long-Term Research in Environmental

Biology (LTREB) program at the National Science Foundation (Grant

Nos DEB-0417678 and DEB-1019522) to William Peterjohn Colin

Averill was supported by the NOAA Climate and Global Change

Postdoctoral Fellowship Program administered by Cooperative Pro-

grams for the Advancement of Earth System Science (CPAESS)

University Corporation for Atmospheric Research (UCAR) Boulder

Colorado USA We acknowledge the WVU Genomics Core Facility

Morgantown WV for support provided to help make this publication

possible We also thank Leah Baldinger Brittany Carver Mark Burn-

ham Hannah Hedrick Jennifer Mangano and Catherine Sesa for

assistance in the field and in the laboratory

ORCID

Joseph E Carrara httporcidorg0000-0003-0597-1175

Colin Averill httporcidorg0000-0003-4035-7760

REFERENCES

Adams M B amp Angradi T R (1996) Decomposition and nutrient

dynamics of hardwood leaf litter in the Femow Whole-Watershed

Acidification Experiment Forest Ecology and Management 1127 61ndash

69 httpsdoiorg1010160378-1127(95)03695-4

Adams M B Burger J Zelazny L amp Baumgras J (2004) Descrip-

tion of the fork mountain long-term soil productivity study Site

characterization USDA Forest Service General Technical Report NE

43 323

Adams M B Edwards P J Wood F amp Kochenderfer J N (1993)

Artificial watershed acidification on the Fernow Experimental Forest

USA Journal of Hydrology 150 505ndash519 httpsdoiorg101016

0022-1694(93)90123-Q

Allison S D Wallenstein M D amp Bradford M A (2010) Soil-carbon

response to warming dependent on microbial physiology Nature Geo-

science 3 336ndash340 httpsdoiorg101038ngeo846

Bae K Fahey T J Yanai R D amp Fisk M (2015) Soil nitrogen avail-

ability affects belowground carbon allocation and soil respiration in

northern hardwood forests of new hampshire Ecosystems 18 1179ndash

1191 httpsdoiorg101007s10021-015-9892-7

Brzostek E R Dragoni D Brown Z A amp Phillips R P (2015) Mycor-

rhizal type determines the magnitude and direction of root-induced

changes in decomposition in a temperate forest New Phytologist

206 1274ndash1282 httpsdoiorg101111nph13303

Brzostek E R amp Finzi A C (2011) Substrate supply fine roots and

temperature control proteolytic enzyme activity in temperate forest

soils Ecology 92 892ndash902 httpsdoiorg10189010-18031

Brzostek E R Fisher J B amp Phillips R P (2014) Modeling the carbon

cost of plant nitrogen acquisition Mycorrhizal trade-offs and multi-

path resistance uptake improve predictions of retranslocation Journal

of Geophysical Research Biogeosciences 119 1684ndash1697

Brzostek E R Greco A Drake J E amp Finzi A C (2013) Root carbon

inputs to the rhizosphere stimulate extracellular enzyme activity and

increase nitrogen availability in temperate forest soils Biogeochem-

istry 115 65ndash76 httpsdoiorg101007s10533-012-9818-9

Burnham M B Cumming J R Adams M B amp Peterjohn W T

(2017) Soluble soil aluminum alters the relative uptake of mineral

nitrogen forms by six mature temperate broadleaf tree species

possible implications for watershed nitrate retention Oecologia

185 1ndash11

Caporaso J G Kuczynski J Stombaugh J Bittinger K Bushman F

D Costello E K Huttley G A (2010) QIIME allows analysis of

high-throughput community sequencing data Nature Methods 7

335ndash336 httpsdoiorg101038nmethf303

Carreiro M M Sinsabaugh R L Repert D A amp Parkhurst D F

(2000) Microbial enzyme shifts explain litter decay responses to sim-

ulated nitrogen deposition Ecology 81 2359ndash2365 httpsdoiorg

1018900012-9658(2000)081[2359MESELD]20CO2

Cheeke T E Phillips R P Brzostek E R Rosling A Bever J D amp

Fransson P (2017) Dominant mycorrhizal association of trees alters

carbon and nutrient cycling by selecting for microbial groups with

distinct enzyme function New Phytologist 214 432ndash442 httpsdoi

org101111nph14343

Cheng W Parton W J Gonzalez-Meler M A Phillips R Asao S

McNickle G G Jastrow J D (2014) Synthesis and modeling

perspectives of rhizosphere priming New Phytologist 201 31ndash44

httpsdoiorg101111nph12440

Comas L H Callahan H S amp Midford P E (2014) Patterns in root

traits of woody species hosting arbuscular and ectomycorrhizas

Implications for the evolution of belowground strategies Ecology and

Evolution 4 2979ndash2990 httpsdoiorg101002ece31147

Datta R Kelkar A Baraniya D Molaei A Moulick A Meena R S amp

Formanek P (2017) Enzymatic degradation of lignin in soil A

review Sustainability 9 1163 httpsdoiorg103390su9071163

CARRARA ET AL | 11

fertilized watershed at the Fernow Both watersheds have stands

of similar age (~45 years old) and tree species assemblages Trees

gt25 cm in diameter at breast height (DBH) were cut in the

N-fertilized watershed (Watershed 3) in 1970 and recovered natu-

rally until 1989 when N treatments began (Peterjohn Adams amp

Gilliam 2017) N is added by aerial fertilization via helicopter at a

rate of 35 kg N ha1 year1 in the form of solid pellet (NH4)2

SO4 (Adams Edwards Wood amp Kochenderfer 1993) The

N-fertilized watershed is 343 ha with a south-facing aspect and

elevation ranging from 735 to 860 m (Gilliam et al 2016) The

reference watershed (Watershed 7) was clear cut from 1963 to

1967 (the upper half in 19631964 and the lower half in

19661977) and kept barren with herbicides until 1969 when it

began natural recovery (Kochenderfer 2006) This watershed is

240 ha with an east-facing aspect and elevation ranging from

731ndash850 m (Gilliam et al 2016) The watersheds are dominated

by arbuscular mycorrhizal associated (AM) tree species that include

tulip poplar (Liriodendron tulipifera) red maple (Acer rubrum) black

cherry (Prunus serotina) and sugar maple (Acer saccharum) which

together account for 59 of the basal area in the reference

watershed and 72 in the N-fertilized watershed Less abundant

ectomycorrhizal associated (ECM) tree species include sweet birch

(Betula lenta) red oak (Quercus rubra) and American beech (Fagus

grandifolia) which together account for 25 and 16 of the total

basal area in the reference and N-fertilized watershed

respectively

22 | Soil sampling protocol

We sampled soils on three dates in June July and August of 2015

in order to capture early peak and late growing season dynamics In

each watershed we sampled soils from an existing network of 10

randomly placed 25 9 25 m plots that have similar tree species

composition (Gilliam et al 1996) Three 10 9 10 cm organic horizon

(OH) samples were collected from each plot One OH sample was

used for root metrics and the other two for measures of soil biogeo-

chemistry After OH removal three 5-cm diameter soil cores were

extracted from each plot to a depth of 15 cm All samples were

stored on ice and returned to West Virginia University for further

processing

Within 24 hr of sampling mineral soil samples were separated

into bulk and rhizosphere fractions Rhizosphere soil was opera-

tionally defined as soil that adhered to roots upon removal from the

soil matrix and was carefully subsampled using forceps and homoge-

nized (sensu Phillips amp Fahey 2005) The remaining soil was consid-

ered mineral bulk soil After root and rhizosphere separation the

bulk and OH soil was homogenized by sieving through a 2-mm

mesh A subsample of each soil sample and fraction was stored at

80degC for assays of enzyme activity All fine roots (lt2 mm) were

washed in deionized water and subsequently dried and weighed to

determine standing root biomass Subsamples of roots from each

plot from the final sampling date were used to examine AM colo-

nization and root morphology

23 | Arbuscular Mycorrhizal colonization and rootmorphology

To determine the extent to which N fertilization altered AM colo-

nization we analyzed a subsample comprising ~13 of all roots from

the mineral and OH horizons for AM colonization Roots were

cleared by incubating in 10 potassium hydroxide for 48ndash96 hr at

room temperature depending on the pigmentation of the roots

Excess pigment was leached from roots by incubating in 85 etha-

nol for 24ndash48 hr This step was necessary due to the dark pigments

present in the woody tissue of our samples Any remaining pigment

was removed by soaking roots in ammoniahydrogen peroxide solu-

tion for 15 min Roots were prepared for staining by acidifying them

in 5 hydrochloric acid and then stained for 5 min in 005 trypan

blue (Comas Callahan amp Midford 2014)

We calculated AM colonization for each sample using the grid-

intersect method (Giovannetti amp Mosse 1980 McGonigle Miller

Evans Fairchild amp Swan 1990) Each sample was suspended in

water on a 1 9 1 cm gridded Petri dish and examined under a

stereoscopic microscope At each root-gridline intersect colonization

was determined by the presence of arbuscules or hyphae Percent

AM colonization was then calculated as the percent of total inter-

sects examined that had visible AM structures Given that roots with

high levels of AM colonization are likely to be more involved in

releasing C to rhizosphere microbes (Kaiser et al 2015) we used

the level of colonization by AM fungi as a metric of the degree to

which roots and microbes were actively coupled

We created digital images of root morphology using an LA2400

desktop scanner (WinRhizo Regent Instruments Inc) and then used

WinRhizo software to determine root length surface area average

diameter and number of forks of each sample All measurements

were expressed per g of root mass and scaled to a per m2 basis

based on total standing root biomass (gm2) at the plot level

(Table 1)

24 | Extracellular enzyme activity

For all soil fractions at each sampling date we assayed the potential

activity of hydrolytic enzymes that release nitrogen (N-Acetylgluco-

saminidase NAG) phosphorus (acid phosphatase AP) and simple

carbon (b-glucosidase BG) Given extracellular enzyme activity is the

rate-limiting step of the decomposition of organic matter we con-

sider these measurements a metric of decomposition (Schimel amp

Weintraub 2003) Soil extracellular enzyme activity has been linked

to mass-loss rates of wood (Sinsabaugh et al 1992 1993) and soil

C-mineralization (Brzostek Greco Drake amp Finzi 2013 Hernandez

amp Hobbie 2010) After thawing 1 g of each sample was homoge-

nized in 50 mM sodium acetate buffer (pH 50) Hydrolytic enzyme

activities were determined using a fluorometric microplate assay

with methylumbelliferone-linked substrates In addition we assayed

for activities of liginolytic oxidative enzymes phenol oxidase and

peroxidase using a colorimetric microplate assay based on the

hydrolysis of L-34-dihydroxyphenylalanine linked substrates (Saiya-

CARRARA ET AL | 3

Cork et al 2002) These assays were performed under saturated

substrate conditions wherein the concentration of enzyme limits

activity

We measured ambient proteolytic activity of each soil sample

following a protocol adapted from Brzostek and Finzi (2011) Each

subsample (2ndash3 g) received 10 ml of 005 M sodium acetate buffer

(pH 50) and 400 ll toluene to inhibit microbial reuptake of amino

acids Initial samples were immediately terminated by adding 3 ml of

trichloroacetic acid Incubated samples were shaken at room temper-

ature for 4 hr prior to termination Proteolysis was expressed as the

difference between incubated and initial amino acid concentrations

Amino acid concentrations were measured following the fluorometric

OPAME method (Jones Owen amp Farrar 2002) where fluorescence

of each sample was a function of amino acid concentration calcu-

lated from a glycine standard curve In contrast to the other enzyme

assays described above this proteolytic enzyme assay is run at ambi-

ent substrate conditions and reflects the limitation of activity by

both enzyme and substrate concentrations

25 | Bacterial and fungal community composition

Soil microbial DNA was extracted from samples collected in July 2015

using the Zymo soil microbe mini prep kit (Zymo Research Corpora-

tion Irvine CA USA) and quantified on a Nanodrop 3300 (Thermo

Scientific Wilmington DE USA) Due to logistical constraints we

chose the July sampling date in order to examine microbial community

composition at the peak of the growing season The fungal ITS1 and

bacterial 16S loci were chosen for amplification via polymerase chain

reaction (PCR) in order to delineate microbial community composition

and the corresponding shifts associated with N amendment All PCR

reactions contained 1ndash5 ng template DNA 19 Mg-included Taq

buffer 08 mM dNTPs 005 U of Taq polymerase (Denville Scientific

South Plainfield NJ USA) and 04 pM each of either ITS1-F_KYO1

(Toju Tanabe Yamamoto amp Sato 2012) and ITS2 primers (Gardes amp

Bruns 1993 White Bruns Lee amp Taylor 1990) for fungal amplifica-

tion or S-D-Bact-0341-b-D-17 and S-D-Bact-0785-a-A-21 (Klind-

worth et al 2013) for bacterial analysis Primers were modified to

include 197 unique 8 bp index sequences (for individual sample ldquobar-

codingrdquo) in addition to Illumina adapters and Nextera sequencing

motifs (sequences available upon request) Reaction conditions were

as follows 95degC denaturation for 3 min 30 cycles of 98degC for 20 s

61degC for 30 s 72degC for 30 s followed by a final extension at 72degC for

5 min All amplification products were resolved on a 1 agarose gel

extracted with a sterile razor and purified using the PureLink Quick

Gel Extraction Kit (Invitrogen Carlsbad CA USA) Purified products

were quantified via Qubit pooled in equimolar amounts and

sequenced in concert on the Illumina MiSeq at the West Virginia

University Genomics Core Facility (Morgantown WV)

Paired end sequences were merged and barcodes and primers

trimmed using the Quantitative insights into Microbial Ecology

(QIIM) pipeline version 190 (Caporaso et al 2010) Using a Phred

score cut-off of 30 reads with less than 75 high quality base calls

were excluded from further analysis We also discarded sequences

with three or more adjacent low-quality base calls Sequences were

clustered into operational taxonomic units (OTUs) using the

USEARCH algorithm with an open reference OTU picking strategy

using a 97 sequence similarity threshold (Edgar 2010) Bacterial

16S sequences were clustered against the Greengenes reference

database (DeSantis et al 2006) and fungal sequences were clus-

tered against the UNITE ITS database (K~oljalg et al 2013) Taxon-

omy was assigned using the RDP 9 22 classifier (Wang Garrity

Tiedje amp Cole 2007) and associated Greengenes and UNITE

Fine root metric Control +N Change

Organic horizon

Root biomass (gmsup2) 3083 407 5213 850 691

Mycorrhizal colonization () 728 36 609 001 163

Specific root length (cmg) 203869 26099 199299 17960 224

Surface area (cmsup2g) 5188 532 8563 2822 251

Average diameter (mm) 044 002 043 002 42

Root volume (cmsup3g) 185 010 185 017 024

Forksg 6027 739 5967 910 098

Mineral soil

Root biomass (gmsup2) 31923 2631 24051 4820 247

Mycorrhizal colonization () 753 58 332 67 559

Specific root length (cmg) 124057 9598 97658 9393 213

Surface area (cmsup2g) 16713 881 12525 847 251

Average diameter (mm) 044 002 043 002 65

Root volume (cmsup3g) 184 012 130 057 294

Forksg 2598 351 2091 183 195

Mean values of all sampling dates are accompanied by standard error

Significant differences between treatments (p lt 05)

TABLE 1 Fine root and mycorrhizalmetrics in bulk and organic horizon soilsunder ambient and long-term N-fertilizedconditions

4 | CARRARA ET AL

reference databases To determine the effect of N fertilization on

overall species composition the top four abundant fungal phyla

(Ascomycota Basidiomycota Chytridiomycota and Zygotmycota as

well as lsquounclassifiedrsquo) were examined for shifts in relative abundance

To further analyze fungal and bacterial community composition

each OTU table was proportionally normalized such that the per OTU

sequence count within a sample was divided by the total number of

sequences detected in a given sample generating a proportion on the

interval [01] This proportional normalization has been demonstrated

to be sufficiently accurate when using the BrayndashCurtis similarity met-

ric (Weiss et al 2017) We then used normalized OTU tables to calcu-

late BrayndashCurtis similarity using the vegdist function within the vegan

package for R statistical software (Oksanen et al 2015 R Core Team

2017) Community similarity was analyzed as a function of watershed

soil horizon and their interaction using the adonis function within the

vegan package If interactions were not significant then they were

removed and we only analyzed main effects

To understand how microbial community composition related to

variation in soil enzyme profiles we created similarity matrices of

soil enzymes We calculated BrayndashCurtis similarity of enzyme profiles

using all enzymes using the vegdist function in vegan We then per-

formed nonmetric multidimensional scaling (NMDS) to generate

NMDS scores for both microbial and enzymatic profiles We tested

for relationships between microbial communities and enzymatic pro-

files in bulk and rhizosphere soil separately by comparing the first

NMDS axes of microbial and enzymatic profiles using linear regres-

sion in SAS JMP vs 1002 (SAS Institute Cary NC USA) We fur-

ther examined the relationship between microbial communities and

enzymatic profiles across all soil horizons by comparing the first

NMDS axes of microbial and enzymatic profiles using linear regres-

sion across both watersheds

To assess the relative importance of belowground C allocation

(ie AM colonization) on fungal and bacterial community composi-

tion we explored whether there were correlations between AM

colonization and the first NMDS axis of bacterial and fungal commu-

nity composition in bulk and rhizosphere soil separately using SAS

JMP vs 1002 (SAS Institute Cary NC USA)

26 | Net mineralization and nitrification

For all soil fractions at each sampling date we measured the rate of

net N mineralization and net nitrification within 48 hours of collec-

tion Net N mineralization and nitrification rates were expressed as

the difference in the 1 M KCl extractable pool sizes of NHthorn4 and

NO3 between an initial sample and a sample that was aerobically

incubated in the lab for 14 days at room temperature and under

constant soil moisture (Finzi Van Breemen amp Canham 1998) To

perform the extractions 50 ml of 1 M KCl was added to a jar con-

taining 5 g of each soil sample Samples were sealed and shaken for

30 min and allowed to settle for 2 hr Samples were filtered using

Pall Supor 450 filters via syringe into 20-ml vials and stored at

20degC prior to analysis by flow injection (Lachat QuikChem 8500

series 2)

27 | Statistical analysis

To determine the extent to which N fertilization altered enzyme

activities N mineralization nitrification and root metrics we per-

formed a three-way analysis of variance (ANOVA) in SAS JMP vs

1002 (SAS Institute Cary NC USA) and used date watershed and

soil horizon as factors Although we included date in our statistical

models we present growing season means in the results reflecting

our focus on treatment rather than seasonal effects Post hoc multi-

ple comparisons were also made among watersheds dates and soil

fractions using the TukeyndashKramer HSD test

28 | Modeling potential impacts of plantndashmicrobiallinkages on soil decomposition

To further examine the extent to which linkages between plants and

soil microbes drive soil enzyme responses to N fertilization as well

as to estimate potential impacts on soil C stocks we used a micro-

bial enzyme decomposition model (Allison Wallenstein amp Bradford

2010) that was modified in Cheng et al (2014) to include seasonal

differences in soil temperature the inputs of root and leaf litter and

root transfers of C to the rhizosphere For leaf and root litter inputs

we used the mean observed litterfall rate from 1998 to 2015 for

each watershed (Kochenderfer 2006 Peterjohn et al 2017) and our

own measurements of root biomass with the assumption that roots

in both watersheds turnover once each year (McCormack Adams

Smithwick amp Eissenstat 2017) For both roots and leaves we

assumed that turnover primarily occurred during the leaf senescence

in the fall It is important to note there are no significant differences

in leaf litterfall rates between the two watersheds (WS3 = 143 g C

m2 WS7 = 146 g Cm2 Kochenderfer 2006) Finally we used pub-

lished values of AM root exudation rates on a per g root basis (Yin

Wheeler amp Phillips 2014) to estimate root C transfers assuming

that these inputs occurred only during the growing season Using

these model inputs we then ran two scenarios (1) the fertilized and

control watersheds had similar root C exudation rates per g of root

and (2) N fertilization reduced exudations rates by 25 We chose

this reduction in exudation rates to be conservative as it represents

around half of the decline in AM colonization we observed in the

fertilized watershed All model simulations were run for 30 year to

achieve steady state

3 | RESULTS

31 | Fine root metrics

Fine root biomass and the extent of mycorrhizal symbiosis in the

bulk mineral soil layer were significantly lower in the N-fertilized

watershed (Table 1) Overall the bulk mineral soil in the N-fertilized

watershed had 247 less standing fine root biomass than the refer-

ence watershed This reduction in fine root biomass was accompa-

nied by decreases in fine root surface area volume and the amount

of root forks per g Additionally N fertilization reduced AM

CARRARA ET AL | 5

colonization of fine roots by 559 Given the inherent limitations of

measuring root production using minirhizotron tubes or ingrowth

cores we use fine root biomass and AM colonization as proxies for

plant C allocation to gain nutrients While root turnover may be

enhanced under N fertilization we base the assumption that fine

root biomass is synonymous with belowground C allocation to fine

roots on our observations that fine root biomass was consistently

lower in the fertilized than control watersheds over all three of the

sequentially cored soil sampling dates during the growing season In

addition AM fungi have been shown to act as conduits for photo-

synthate C to rhizosphere microbes and as such represent an impor-

tant belowground flux of C (Kaiser et al 2015) Thus reduced

investment in AM colonization may also lead to reduced transfers of

photosynthate C to the rhizosphere resulting in an overall decline in

belowground C allocation

By contrast in the OH standing fine root biomass was larger in

the N-fertilized watershed although root biomass in the OH horizon

is markedly lower than in the bulk mineral soil (Table 1) However

like the bulk mineral soil AM colonization of OH roots was signifi-

cantly higher in the reference watershed (728 colonized) than the

N-fertilized watershed (609 colonized)

32 | Extracellular enzyme activity

Nearly all hydrolytic enzyme activities were lower under elevated N

conditions across all three soil fractions BG activity was significantly

lower in the N-fertilized watershed in both the bulk mineral (52

decrease) and OH (17 decrease) soil fractions (p lt 05 Figure 1a)

N fertilization significantly lowered AP activity in the bulk (12) and

rhizosphere (36) fractions of the mineral soil (p lt 05 Figure 1b)

Similarly N amendment significantly lowered NAG activity in both

bulk and rhizosphere fractions of the mineral soil by 41 and 37

respectively (p lt 05 Figure 1c) However BG activity in the rhizo-

sphere of the mineral soil was not significantly different between

0

04

08

12

16

Bulk Rhizo

AP

(m

olmiddot[

gdr

yso

il]ndash1

middot hr

ndash1)

0

1

2

3

4

5

OH0

01

02

03

04

Bulk Rhizo

BG

(m

olmiddot[g

dry

soi

l]ndash1

middot hr

ndash1)

0

05

1

15

2

OH

0

003

006

009

012

Bulk Rhizo

NA

G (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)

0

02

04

06

08

1

OH

0

04

08

12

16

Bulk Rhizo0

10

20

30

40

50

OH

0

025

05

075

1

Bulk Rhizo0

1

2

3

OH

0

1

2

3

4

5

Bulk Rhizo

Pero

xida

se (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)

0

1

2

3

4

OH

(a) (b)

(c) (d)

(e) (f)

Phen

ol o

xida

se (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)Pr

oteo

lysi

s (

gA

A-N

middot[g

dry

soil]

ndash1 middot

hrndash1

)

Reference

+N

F IGURE 1 N fertilization reduceshydrolytic and oxidative enzyme activitiesValues are the overall seasonal meanenzyme activities (mean SE) of (a) BG(b) AP (c) NAG (d) phenol oxidase (e)peroxidase and (f) proteolysis for each soilfraction (ie bulk rhizosphere and organichorizon) measured in June July andAugust 2015 across all plots (n = 10 plotsper watershed) Asterisks indicatesignificant differences in enzyme activitybetween watersheds within soil fractions(p lt 05) Note difference in scale betweenOH vs rhizosphere and bulk soil fractionsAA-N is amino acid nitrogen

6 | CARRARA ET AL

watersheds In addition AP and NAG activities in the OH horizon

did not vary by treatment

Liginolytic oxidative enzyme activities were also generally lower

in the N-fertilized watershed Phenol oxidase activity was signifi-

cantly lower in bulk (45 decrease) rhizosphere (49 decrease) and

OH horizons (57 decrease) of the fertilized watershed relative to

the reference watershed (p lt 05 Figure 1d) Peroxidase activity was

lower in the fertilized watershed in the bulk rhizosphere and OH

soil fractions with reductions of 30 25 and 36 respectively

(p lt 05 Figure 1e) Proteolytic enzyme activity was consistently

lower in each soil fraction under N fertilization with a 48 decrease

in bulk 56 decrease in rhizosphere and 40 decrease in OH

proteolysis (Figure 1f)

33 | Microbial community composition

When fungal taxonomic units were aggregated to the phylum level

there were no significant changes in the relative abundance of the

four most common fungal phyla or the unclassified group in any soil

horizon (Figure S1) When bacterial taxonomic units were aggregated

to the phylum level there were limited shifts in the relative abun-

dance of the seven most common bacterial phyla Relative abun-

dance of Actinobacteria was higher in the OH soil of the fertilized

watershed (Figure S2a) Relative abundance of Proteobacteria was

lower in the fertilized bulk soil and relative abundance of Firmicutes

was higher in both rhizosphere and bulk soil in the fertilized water-

shed (Figure S2bc)

Adonis analysis of bacterial communities revealed significant

effects for watershed soil horizon (p lt 001) and their interaction

(p = 02 total model R2 = 32) Within watersheds post hoc compar-

isons showed OH communities were different than bulk and rhizo-

sphere communities (Figure S3ab) There was no difference

between bulk and rhizosphere communities (Figure S3ab) Across

watersheds bacterial community composition differed in all soil frac-

tions such that OH rhizosphere and bulk soil exhibited unique com-

munities in the N-fertilized watershed compared to the reference

(Figure 2ace)

Adonis analysis of fungal communities revealed significant effects

of watershed and soil horizon (p lt 001 total model R2 = 12) but

not their interaction Within watersheds post hoc comparisons of

fungal communities showed OH communities were different than

bulk and rhizosphere communities in both watersheds but there was

no difference between bulk and rhizosphere communities

(Figure S3cd) Across watersheds fungal community composition

within OH and bulk soil fractions was significantly different (p lt 01

Figure 2bd) However there was no significant difference in fungal

communities between watersheds in rhizosphere soil (Figure 2f)

Comparison of bacterial and enzymatic NMDS scores across all

soil horizons showed a positive relationship between bacterial com-

munities and enzyme profiles (R2 = 48 p lt 01 Figure S4a) Compar-

ison of fungal and enzymatic NMDS scores across all soil horizons and

both watersheds showed no significant relationship between fungal

communities and enzyme profiles (Figure S4b) Across watersheds

comparison of bacterial and enzymatic NMDS scores showed a nega-

tive relationship between bacterial communities and enzyme profiles

in the bulk soil (R2 = 36 p lt 05 Figure 3a) but no significant rela-

tionship in rhizosphere soil Comparison of fungal and enzymatic

NMDS scores across watersheds showed no significant relationships

in either bulk or rhizosphere soil (Figure 3b) Linear regression of

AM colonization and the first bacterial NMDS axis resulted in a posi-

tive linear relationship in bulk soil (R2 = 063 p lt 001 Figure 3c) but

not rhizosphere soil There were no significant linear relationships

between AM colonization and the first fungal NMDS axis in either

soil fraction (Figure 3d)

34 | Net N mineralization and nitrification

The only significant difference we found in rates of nitrogen cycling

was that N mineralization and net nitrification in the OH were 40

and 51 higher in the fertilized watershed than the reference N

transformation rates did not significantly differ between watersheds

in either the bulk or rhizosphere soil fractions (Figure 4ab)

35 | Modeling potential impacts of plantndashmicrobiallinkages on soil decomposition

When we ran the model to steady state we found that reductions

in root biomass and root C transfers to the rhizosphere in the N-fer-

tilized watershed reduced microbial enzyme pools by ~16 and

enhanced soil C by ~3 compared to the reference watershed

(Table 2) Even though exudation rates on a per g root basis were

the same the fertilized watershed had lower overall exudations rates

at the ecosystem scale than the control watershed because of its

lower root biomass (Table 2) When we reduced exudation rates by

25 on a per g root basis in the fertilized watershed there was a

further exacerbation in the reduction in microbial enzyme activity to

a ~28 decline and a larger increase in soil C by ~20 (Table 2)

4 | DISCUSSION

Here we provide evidence that coupled interactions among plants

fungi and bacteria play an important role in enzyme activity

responses to N fertilization For fungi we observed distinct shifts in

fungal community structure in response to N fertilization (Figure 2b

d) but we found no evidence for a link between these shifts and

extracellular enzyme activity (Figure 3b) By contrast we found that

bacterial community composition shifts under N fertilization are cor-

related with declines in enzyme activity (Figure 3a) and that these

compositional shifts are tightly coupled to reductions in plant C allo-

cation to roots and mycorrhizal fungi (Figure 3c) Overall these

results suggest that N fertilization drives an integrated ecosystem

response whereby reductions in plant C allocation to roots and AM

fungi feedback on bacterial community structure and function

While whole-watershed fertilization at the Fernow results in a

pseudoreplicated experimental design (Hurlbert 1984) we conclude

CARRARA ET AL | 7

that the effects we measured are driven by N fertilization rather

than pre-existing differences between these adjacent watersheds

for four main reasons First soil chemistry (ie soil pH cation

exchange capacity nutrient content etc) were similar at the begin-

ning of the experiment (Adams amp Angradi 1996) Second the

amount of N added to the watershed yearly was originally chosen in

1989 to approximately double ambient N deposition rates but is

now more than quadruple current rates and as such it seems unli-

kely that this does not incur a biogeochemical response Third the

results from this watershed study are consistent with measurements

we made during the same year in a replicated N fertilization study

lt2 km away from these watersheds (the Fork Mountain Long-Term

Soil Productivity Plots Adams Burger Zelazny amp Baumgras 2004)

In this small-scale replicated study N fertilization reduced fine root

biomass and ligninolytic enzyme activity (Figure S5) Finally this

work builds upon other research at the Fernow that has found the

N-fertilized watershed had lower rates of litter decomposition

(Adams amp Angradi 1996) reduced understory richness (Gilliam et al

1994 2016 Walter Adams Gilliam amp Peterjohn 2017) and altered

N cycling (Adams et al 1993 Burnham Cumming Adams amp Peter-

john 2017 Gilliam Yurish amp Adams 2001 Gilliam et al 1996

2016) compared to the reference watershed

While we did observe significant declines in enzyme activity

across all three soil fractions particularly for the liginolytic enzymes

(Figure 1andashf) these declines were not correlated with significant

shifts in fungal community composition (Figure 3b) The lack of a

clear link between enzyme declines and changes in the fungal com-

munity does not support the prevailing paradigm that white-rot

Basidiomycota are the dominant cause of a decline in enzyme activi-

ties following N additions (Edwards Zak Kellner Eisenlord amp Pregit-

zer 2011 Fog 1988 Freedman Romanowicz Upchurch amp Zak

2015 Morrison et al 2016) It is possible that fungal enzyme activ-

ity response to N fertilization is independent of community composi-

tion (ie investment in enzyme activity declines with no change in

community structure) however microbial community composition

has been linked to catabolic functioning and enzyme activities across

N gradients and seasons (Fierer et al 2011 Vorıskova Brabcova

Cajthaml amp Baldrian 2014) Furthermore our data indicate that

ndash008

0

008

016

024

032

ndash09 ndash06 ndash03 0 03 06

Rhi

zo f

ungi

NM

DS2

Rhizo fungi NMDS1

ndash04

ndash02

0

02

04

ndash02 ndash01 0 01 02 03 04

Rhi

zo b

acte

ria

NM

DS2

Rhizo bacteria NMDS1

ndash02

ndash01

0

01

02

03

ndash02 0 02 04 06

Bul

k ba

cter

ia N

MD

S2

Bulk bacteria NMDS1

ndash04

ndash032

ndash024

ndash016

ndash008

0

ndash04 ndash02 0 02 04

OH

fun

gi N

MD

S2

OH fungi NMDS1

ndash06

ndash04

ndash02

0

02

ndash05 ndash04 ndash03 ndash02 ndash01 0

OH

bac

teri

a N

MD

S2

OH bacteria NMDS1

Reference

+N

ndash02

ndash01

0

01

02

03

04

ndash06 ndash04 ndash02 0 02 04

Bul

k fu

ngi N

MD

S2

Bulk fungi NMDS1

(a) (b)

(c) (d)

(e) (f)

plt01

plt01

plt01

plt01

plt01

F IGURE 2 N fertilization alteredbacterial community composition in OH (a)bulk (c) and rhizosphere (e) soils andfungal community composition in OH (b)and bulk (d) soils but not rhizosphere (f)All community data were obtained for eachsoil fraction in July 2015 (n = 10 plots perwatershed) p values indicate significantdifferences between the N-fertilized andreference community

8 | CARRARA ET AL

shifts in bacterial community composition under elevated N are cor-

related with reduced enzyme activity at the Fernow (Figure 3a)

While changes in bacterial community may be influenced by overall

fungal community composition these shifts in bacterial community

composition are tightly coupled to AM colonization (a metric of

belowground C allocation) suggesting that plant responses to N fer-

tilization feedback on bacterial community structure and function

(Figure 3c) While we cannot rule out that N-induced declines in

total fungal biomass led to reductions in liginolytic enzyme activity

(DeForest et al 2004 Frey et al 2004 Treseder 2008 Wallenstein

et al 2006) the dominance of AM trees in our plots whose inor-

ganic nutrient economy is largely driven by bacteria suggests that

free-living fungi may not be an important driver of N deposition

responses in AM-dominated systems (Cheeke et al 2017 Phillips

Brzostek amp Midgley 2013)

In contrast it appears that the declines in enzyme activity we

observed appear to be the result of a cascade of ecosystem

responses affecting both microbial community composition and plant

C allocation belowground The N-induced declines in fine root bio-

mass AM colonization and root morphology all indicate that there

was a reduction in the investment of C belowground by trees to gain

nutrients (Table 1) While previous research has shown that below-

ground C allocation is inversely correlated with N availability (Bae

Fahey Yanai amp Fisk 2015 Haynes amp Gower 1995 Phillips amp Fahey

2005) the tight linkage between the first bacterial NMDS axis and

AM colonization suggest that N-induced declines in belowground

C allocation caused a shift in the bacterial community composition

and subsequent reductions in enzyme activity (Figure 3ac) This

result also supports the emerging view that roots are active engi-

neers of microbial activity (Brzostek et al 2013 Finzi et al 2015

Yin et al 2014) and highlights the need to integrate root-microbial

interactions into our conceptual and predictive modeling frameworks

of how forest ecosystems respond to global change (Johnson

Gehring amp Jansa 2016 Terrer Vicca Hungate Phillips amp Prentice

2016)

An additional impact of the N fertilization treatment is that it

reduced soil pH (as measured in a 001 M calcium chloride buffer) of

the upper 5 cm of mineral soil in the fertilized watershed to 34

compared to 38 in the control watershed (Peterjohn unpublished

data) Soil pH is an important control on microbial community diver-

sity (Fierer et al 2012 Kaiser et al 2016 Lauber Hamady Knight

amp Fierer 2009) microbial biomass and enzyme activities (Rousk amp

Baath 2011 Sinsabaugh 2010) Thus the decline in soil pH may

account for a portion of the enzyme reductions we observed under

N fertilization To address this we assayed the sensitivity of phenol

oxidase and peroxidase enzyme activity in organic horizon bulk and

rhizosphere soils from the control watershed to three different levels

of pH that spanned a 1 unit shift (data not shown) Peroxidase activ-

ity was insensitive to pH (slope = 074 r2 = 05 p gt 05) While

phenol oxidase activity did significantly increase as pH increased

(slope = 010 r2 = 053 p lt 05) this sensitivity would only account

for a 10 decline in activity Given that we observed a 50 decline

in phenol oxidase in the treatment watershed coupled with the

greater overall importance of peroxidase enzymes in our study (ie

nearly an order of magnitude higher activity) it appears that pH is

an important but secondary driver of the enzyme activity responses

we observed

Over longer time scales the reductions in root and microbial

activity we observed at the Fernow may have important implications

for soil C stocks In our model simulation we found that feedbacks

between reductions in root biomass and enzyme production have

the potential to drive nearly a 3 increase in soil C stocks (Table 2)

When these were coupled with a 25 reduction in specific root C

exudation rates soil C in the fertilized watershed increased by nearly

20 over the 30-yr simulation This model was designed to be theo-

retical and as such it is used here to show the sensitivity of the

y = 0004x ndash 007R = 63

ndash02

0

02

04

06

0 25 50 75 100Bul

k B

acte

ria

OT

U N

MD

S1

AM Colonization ()

y = ndash0159x + 013R = 360

004

008

012

016

02

ndash02 0 02 04 06

Enz

yme

NM

DS

1

Bacteria OTU NMDS1

+NReference

0

004

008

012

016

02

ndash06 ndash04 ndash02 0 02 04

Enz

yme

NM

DS

1

Fungal OTU NMDS1

ndash06

ndash04

ndash02

0

02

04

0 25 50 75 100

Bul

k Fu

ngi

OT

U N

MD

S1

AM Colonization ()

plt05

plt001

(a)

(c)

(b)

(d)F IGURE 3 Bacterial but not fungalcommunity composition is correlated withthe first NMDS axis of enzyme activity (aamp b) Percent AM colonization is correlatedwith bacterial community composition (c)but not fungal community composition (d)Data presented are bulk soil communitycomposition data from July 2015 (n = 10plots per watershed) p values indicatesignificance of correlation

CARRARA ET AL | 9

system to the perturbations in plant inputs and is not meant to be

quantitatively predictive However this simple modeling effort pro-

vides compelling evidence that N-induced shifts in rootndashmicrobial

interactions have the potential to alter not only microbial production

of soil enzymes but also the size of the soil C pool To generate the

necessary data for fully integrating root-microbial interactions into

more sophisticated ecosystem models future research should couple

direct measurements of belowground C allocation responses to N

fertilization (eg total belowground C allocation root exudation)

with the resulting impacts on microbial activity

Given the prevailing paradigm that free-living fungi drive soil

enzyme activity and subsequent decomposition responses to N fer-

tilization (Carreiro et al 2000 Fog 1988 Waldrop et al 2004 Zak

et al 2008) our results raise an important question of why shifts in

plant C allocation bacterial community structure and enzyme activi-

ties were tightly coupled at the Fernow The divergent results

between the Fernow and other long-term fertilization sites may be

the result of differences in ambient N deposition and tree species

composition The Fernow is in a region that has historically received

some of the highest levels of N deposition in the United States

(Driscoll et al 2001) which may have enhanced bacterial dominance

before treatment began in 1989 By contrast N fertilization experi-

ments that have linked reductions in soil C decomposition to fungal

community composition and activity tend to be located in areas with

lower historical N deposition rates (Edwards et al 2011 Freedman

et al 2015 Frey et al 2004 Morrison et al 2016 Wallenstein

et al 2006) Second the Fernow is predominantly dominated by

AM trees whereas most N fertilization experiments have tended to

occur in ecosystems dominated by ECM trees with ECM-dominated

sites comprising ~80 of the studies included in the Janssens et al

(2010) meta-analysis AM trees show greater growth enhancements

with N addition (Thomas et al 2010) are characterized by lower

fungal to bacterial ratios (Cheeke et al 2017) and are less depen-

dent on plantndashmicrobial interactions to access N than ECM trees

(Brzostek Dragoni Brown amp Phillips 2015) As such in AM-domi-

nated systems like the Fernow bacteria may outweigh fungi in con-

trolling soil decomposition responses to N fertilization Additionally

evidence that ECM trees rely more on roots and rhizosphere

microbes to access N than AM trees suggests that N fertilization

may lead to greater declines in belowground C allocation and

enzyme activity than observed at the Fernow (Brzostek et al 2015

Yin et al 2014) Moving forward the equal distribution of ECM and

AM trees across the temperate forest landscape highlights a critical

need to investigate ECM and AM stand responses to N fertilization

within the same ecosystem (Midgley amp Phillips 2014)

Although enzyme declines were observed across all three soil

fractions (Figure 1andashf) N cycling shifts were only observed in the

OH where rates were elevated by nearly 50 and 40 for mineral-

ization and nitrification respectively (Figure 4ab) OH and mineral

soils differ in key biogeochemical traits and this may contribute to

these divergent N cycling responses In temperate forests OH

0

05

1

15

Bulk Rhizo

Net

N m

iner

aliz

atio

n (

gN

middotgndash1

middotday

ndash1)

Control

+N

0

3

6

9

12

15

18

OH

0

05

1

15

Bulk Rhizo

Net

nitr

ific

atio

n ((

gN

middotgndash1

middotday

ndash1)

0

2

4

6

8

10

12

OH

(a)

(b)

Reference

F IGURE 4 N fertilization increases (a) N mineralization and (b)nitrification in the OH but not the mineral soil Values are theoverall watershed-level mean rates (mean SE) measured in JuneJuly and August 2015 for each soil fraction (n = 10 plots perwatershed) Asterisks indicate significant differences betweentreatments (p lt 05)

TABLE 2 At model steady state fertilization reduced microbialenzyme pools by ~16 and enhanced soil C by ~3

Watershed ExudationEnzyme pool(mgcm3)

Soil C pool(mgcm3)

Control No change 00136 18473

Fertilized No change 00114 19092

25 reduction 00097 22116

When root C exudation was reduced 25 on a per g root basis in the

fertilized watershed there was a further exacerbation in the reduction in

microbial enzyme activity to a ~28 decline and a larger increase in soil

C by ~20

10 | CARRARA ET AL

typically have rapid N cycling rates reflecting greater organic matter

content and root densities than mineral soils (Brzostek amp Finzi

2011) Shifts in the ratio of gross immobilization to gross mineraliza-

tion may also play a role Net N mineralization rates increased in the

OH despite N fertilization induced declines in proteolytic and chiti-

nolytic enzyme activity that produce N monomers for microbial

uptake While we do not have data on gross N cycling we hypothe-

size that declines in gross microbial N immobilization due to reduced

N demand may have outpaced declines in gross N mineralization in

the fertilized watershed Regardless of the exact mechanism it is

important to put the OH results into context on a mass per unit

area basis the OH is less important to total ecosystem N cycling

than the underlying mineral soil

Much of the research on soil N fertilization has focused on

aboveground responses such as litter input and quality or fungal

community shifts to explain widely observed reductions in enzyme

activities and subsequent soil decomposition (Edwards et al 2011

Frey et al 2004 Morrison et al 2016 Sinsabaugh et al 2008

Waldrop et al 2004 Wallenstein et al 2006) Our results provide

evidence that enzyme activity declines under long-term N fertiliza-

tion at the Fernow are driven by a cascade of ecosystem responses

whereby reductions in belowground plant C investment lead to

shifts in bacterial community structure and a decline in their ability

to degrade soil organic matter Thus there is a need to integrate

plantndashmicrobial interactions into our current conceptual and predic-

tive models of N deposition impacts on temperate forests in order

to forecast soil C responses to shifting N deposition regimes

ACKNOWLEDGEMENTS

We acknowledge Mary Beth Adams Tom Schuler and the US Forest

Service staff at the Fernow Experimental Forest for logistical assis-

tance and access to the experimental watersheds and the LTSP site

This work was also supported by the National Science Foundation

Graduate Research Fellowship to Joseph Carrara under Grant No

DGE-1102689 and by the Long-Term Research in Environmental

Biology (LTREB) program at the National Science Foundation (Grant

Nos DEB-0417678 and DEB-1019522) to William Peterjohn Colin

Averill was supported by the NOAA Climate and Global Change

Postdoctoral Fellowship Program administered by Cooperative Pro-

grams for the Advancement of Earth System Science (CPAESS)

University Corporation for Atmospheric Research (UCAR) Boulder

Colorado USA We acknowledge the WVU Genomics Core Facility

Morgantown WV for support provided to help make this publication

possible We also thank Leah Baldinger Brittany Carver Mark Burn-

ham Hannah Hedrick Jennifer Mangano and Catherine Sesa for

assistance in the field and in the laboratory

ORCID

Joseph E Carrara httporcidorg0000-0003-0597-1175

Colin Averill httporcidorg0000-0003-4035-7760

REFERENCES

Adams M B amp Angradi T R (1996) Decomposition and nutrient

dynamics of hardwood leaf litter in the Femow Whole-Watershed

Acidification Experiment Forest Ecology and Management 1127 61ndash

69 httpsdoiorg1010160378-1127(95)03695-4

Adams M B Burger J Zelazny L amp Baumgras J (2004) Descrip-

tion of the fork mountain long-term soil productivity study Site

characterization USDA Forest Service General Technical Report NE

43 323

Adams M B Edwards P J Wood F amp Kochenderfer J N (1993)

Artificial watershed acidification on the Fernow Experimental Forest

USA Journal of Hydrology 150 505ndash519 httpsdoiorg101016

0022-1694(93)90123-Q

Allison S D Wallenstein M D amp Bradford M A (2010) Soil-carbon

response to warming dependent on microbial physiology Nature Geo-

science 3 336ndash340 httpsdoiorg101038ngeo846

Bae K Fahey T J Yanai R D amp Fisk M (2015) Soil nitrogen avail-

ability affects belowground carbon allocation and soil respiration in

northern hardwood forests of new hampshire Ecosystems 18 1179ndash

1191 httpsdoiorg101007s10021-015-9892-7

Brzostek E R Dragoni D Brown Z A amp Phillips R P (2015) Mycor-

rhizal type determines the magnitude and direction of root-induced

changes in decomposition in a temperate forest New Phytologist

206 1274ndash1282 httpsdoiorg101111nph13303

Brzostek E R amp Finzi A C (2011) Substrate supply fine roots and

temperature control proteolytic enzyme activity in temperate forest

soils Ecology 92 892ndash902 httpsdoiorg10189010-18031

Brzostek E R Fisher J B amp Phillips R P (2014) Modeling the carbon

cost of plant nitrogen acquisition Mycorrhizal trade-offs and multi-

path resistance uptake improve predictions of retranslocation Journal

of Geophysical Research Biogeosciences 119 1684ndash1697

Brzostek E R Greco A Drake J E amp Finzi A C (2013) Root carbon

inputs to the rhizosphere stimulate extracellular enzyme activity and

increase nitrogen availability in temperate forest soils Biogeochem-

istry 115 65ndash76 httpsdoiorg101007s10533-012-9818-9

Burnham M B Cumming J R Adams M B amp Peterjohn W T

(2017) Soluble soil aluminum alters the relative uptake of mineral

nitrogen forms by six mature temperate broadleaf tree species

possible implications for watershed nitrate retention Oecologia

185 1ndash11

Caporaso J G Kuczynski J Stombaugh J Bittinger K Bushman F

D Costello E K Huttley G A (2010) QIIME allows analysis of

high-throughput community sequencing data Nature Methods 7

335ndash336 httpsdoiorg101038nmethf303

Carreiro M M Sinsabaugh R L Repert D A amp Parkhurst D F

(2000) Microbial enzyme shifts explain litter decay responses to sim-

ulated nitrogen deposition Ecology 81 2359ndash2365 httpsdoiorg

1018900012-9658(2000)081[2359MESELD]20CO2

Cheeke T E Phillips R P Brzostek E R Rosling A Bever J D amp

Fransson P (2017) Dominant mycorrhizal association of trees alters

carbon and nutrient cycling by selecting for microbial groups with

distinct enzyme function New Phytologist 214 432ndash442 httpsdoi

org101111nph14343

Cheng W Parton W J Gonzalez-Meler M A Phillips R Asao S

McNickle G G Jastrow J D (2014) Synthesis and modeling

perspectives of rhizosphere priming New Phytologist 201 31ndash44

httpsdoiorg101111nph12440

Comas L H Callahan H S amp Midford P E (2014) Patterns in root

traits of woody species hosting arbuscular and ectomycorrhizas

Implications for the evolution of belowground strategies Ecology and

Evolution 4 2979ndash2990 httpsdoiorg101002ece31147

Datta R Kelkar A Baraniya D Molaei A Moulick A Meena R S amp

Formanek P (2017) Enzymatic degradation of lignin in soil A

review Sustainability 9 1163 httpsdoiorg103390su9071163

CARRARA ET AL | 11

Cork et al 2002) These assays were performed under saturated

substrate conditions wherein the concentration of enzyme limits

activity

We measured ambient proteolytic activity of each soil sample

following a protocol adapted from Brzostek and Finzi (2011) Each

subsample (2ndash3 g) received 10 ml of 005 M sodium acetate buffer

(pH 50) and 400 ll toluene to inhibit microbial reuptake of amino

acids Initial samples were immediately terminated by adding 3 ml of

trichloroacetic acid Incubated samples were shaken at room temper-

ature for 4 hr prior to termination Proteolysis was expressed as the

difference between incubated and initial amino acid concentrations

Amino acid concentrations were measured following the fluorometric

OPAME method (Jones Owen amp Farrar 2002) where fluorescence

of each sample was a function of amino acid concentration calcu-

lated from a glycine standard curve In contrast to the other enzyme

assays described above this proteolytic enzyme assay is run at ambi-

ent substrate conditions and reflects the limitation of activity by

both enzyme and substrate concentrations

25 | Bacterial and fungal community composition

Soil microbial DNA was extracted from samples collected in July 2015

using the Zymo soil microbe mini prep kit (Zymo Research Corpora-

tion Irvine CA USA) and quantified on a Nanodrop 3300 (Thermo

Scientific Wilmington DE USA) Due to logistical constraints we

chose the July sampling date in order to examine microbial community

composition at the peak of the growing season The fungal ITS1 and

bacterial 16S loci were chosen for amplification via polymerase chain

reaction (PCR) in order to delineate microbial community composition

and the corresponding shifts associated with N amendment All PCR

reactions contained 1ndash5 ng template DNA 19 Mg-included Taq

buffer 08 mM dNTPs 005 U of Taq polymerase (Denville Scientific

South Plainfield NJ USA) and 04 pM each of either ITS1-F_KYO1

(Toju Tanabe Yamamoto amp Sato 2012) and ITS2 primers (Gardes amp

Bruns 1993 White Bruns Lee amp Taylor 1990) for fungal amplifica-

tion or S-D-Bact-0341-b-D-17 and S-D-Bact-0785-a-A-21 (Klind-

worth et al 2013) for bacterial analysis Primers were modified to

include 197 unique 8 bp index sequences (for individual sample ldquobar-

codingrdquo) in addition to Illumina adapters and Nextera sequencing

motifs (sequences available upon request) Reaction conditions were

as follows 95degC denaturation for 3 min 30 cycles of 98degC for 20 s

61degC for 30 s 72degC for 30 s followed by a final extension at 72degC for

5 min All amplification products were resolved on a 1 agarose gel

extracted with a sterile razor and purified using the PureLink Quick

Gel Extraction Kit (Invitrogen Carlsbad CA USA) Purified products

were quantified via Qubit pooled in equimolar amounts and

sequenced in concert on the Illumina MiSeq at the West Virginia

University Genomics Core Facility (Morgantown WV)

Paired end sequences were merged and barcodes and primers

trimmed using the Quantitative insights into Microbial Ecology

(QIIM) pipeline version 190 (Caporaso et al 2010) Using a Phred

score cut-off of 30 reads with less than 75 high quality base calls

were excluded from further analysis We also discarded sequences

with three or more adjacent low-quality base calls Sequences were

clustered into operational taxonomic units (OTUs) using the

USEARCH algorithm with an open reference OTU picking strategy

using a 97 sequence similarity threshold (Edgar 2010) Bacterial

16S sequences were clustered against the Greengenes reference

database (DeSantis et al 2006) and fungal sequences were clus-

tered against the UNITE ITS database (K~oljalg et al 2013) Taxon-

omy was assigned using the RDP 9 22 classifier (Wang Garrity

Tiedje amp Cole 2007) and associated Greengenes and UNITE

Fine root metric Control +N Change

Organic horizon

Root biomass (gmsup2) 3083 407 5213 850 691

Mycorrhizal colonization () 728 36 609 001 163

Specific root length (cmg) 203869 26099 199299 17960 224

Surface area (cmsup2g) 5188 532 8563 2822 251

Average diameter (mm) 044 002 043 002 42

Root volume (cmsup3g) 185 010 185 017 024

Forksg 6027 739 5967 910 098

Mineral soil

Root biomass (gmsup2) 31923 2631 24051 4820 247

Mycorrhizal colonization () 753 58 332 67 559

Specific root length (cmg) 124057 9598 97658 9393 213

Surface area (cmsup2g) 16713 881 12525 847 251

Average diameter (mm) 044 002 043 002 65

Root volume (cmsup3g) 184 012 130 057 294

Forksg 2598 351 2091 183 195

Mean values of all sampling dates are accompanied by standard error

Significant differences between treatments (p lt 05)

TABLE 1 Fine root and mycorrhizalmetrics in bulk and organic horizon soilsunder ambient and long-term N-fertilizedconditions

4 | CARRARA ET AL

reference databases To determine the effect of N fertilization on

overall species composition the top four abundant fungal phyla

(Ascomycota Basidiomycota Chytridiomycota and Zygotmycota as

well as lsquounclassifiedrsquo) were examined for shifts in relative abundance

To further analyze fungal and bacterial community composition

each OTU table was proportionally normalized such that the per OTU

sequence count within a sample was divided by the total number of

sequences detected in a given sample generating a proportion on the

interval [01] This proportional normalization has been demonstrated

to be sufficiently accurate when using the BrayndashCurtis similarity met-

ric (Weiss et al 2017) We then used normalized OTU tables to calcu-

late BrayndashCurtis similarity using the vegdist function within the vegan

package for R statistical software (Oksanen et al 2015 R Core Team

2017) Community similarity was analyzed as a function of watershed

soil horizon and their interaction using the adonis function within the

vegan package If interactions were not significant then they were

removed and we only analyzed main effects

To understand how microbial community composition related to

variation in soil enzyme profiles we created similarity matrices of

soil enzymes We calculated BrayndashCurtis similarity of enzyme profiles

using all enzymes using the vegdist function in vegan We then per-

formed nonmetric multidimensional scaling (NMDS) to generate

NMDS scores for both microbial and enzymatic profiles We tested

for relationships between microbial communities and enzymatic pro-

files in bulk and rhizosphere soil separately by comparing the first

NMDS axes of microbial and enzymatic profiles using linear regres-

sion in SAS JMP vs 1002 (SAS Institute Cary NC USA) We fur-

ther examined the relationship between microbial communities and

enzymatic profiles across all soil horizons by comparing the first

NMDS axes of microbial and enzymatic profiles using linear regres-

sion across both watersheds

To assess the relative importance of belowground C allocation

(ie AM colonization) on fungal and bacterial community composi-

tion we explored whether there were correlations between AM

colonization and the first NMDS axis of bacterial and fungal commu-

nity composition in bulk and rhizosphere soil separately using SAS

JMP vs 1002 (SAS Institute Cary NC USA)

26 | Net mineralization and nitrification

For all soil fractions at each sampling date we measured the rate of

net N mineralization and net nitrification within 48 hours of collec-

tion Net N mineralization and nitrification rates were expressed as

the difference in the 1 M KCl extractable pool sizes of NHthorn4 and

NO3 between an initial sample and a sample that was aerobically

incubated in the lab for 14 days at room temperature and under

constant soil moisture (Finzi Van Breemen amp Canham 1998) To

perform the extractions 50 ml of 1 M KCl was added to a jar con-

taining 5 g of each soil sample Samples were sealed and shaken for

30 min and allowed to settle for 2 hr Samples were filtered using

Pall Supor 450 filters via syringe into 20-ml vials and stored at

20degC prior to analysis by flow injection (Lachat QuikChem 8500

series 2)

27 | Statistical analysis

To determine the extent to which N fertilization altered enzyme

activities N mineralization nitrification and root metrics we per-

formed a three-way analysis of variance (ANOVA) in SAS JMP vs

1002 (SAS Institute Cary NC USA) and used date watershed and

soil horizon as factors Although we included date in our statistical

models we present growing season means in the results reflecting

our focus on treatment rather than seasonal effects Post hoc multi-

ple comparisons were also made among watersheds dates and soil

fractions using the TukeyndashKramer HSD test

28 | Modeling potential impacts of plantndashmicrobiallinkages on soil decomposition

To further examine the extent to which linkages between plants and

soil microbes drive soil enzyme responses to N fertilization as well

as to estimate potential impacts on soil C stocks we used a micro-

bial enzyme decomposition model (Allison Wallenstein amp Bradford

2010) that was modified in Cheng et al (2014) to include seasonal

differences in soil temperature the inputs of root and leaf litter and

root transfers of C to the rhizosphere For leaf and root litter inputs

we used the mean observed litterfall rate from 1998 to 2015 for

each watershed (Kochenderfer 2006 Peterjohn et al 2017) and our

own measurements of root biomass with the assumption that roots

in both watersheds turnover once each year (McCormack Adams

Smithwick amp Eissenstat 2017) For both roots and leaves we

assumed that turnover primarily occurred during the leaf senescence

in the fall It is important to note there are no significant differences

in leaf litterfall rates between the two watersheds (WS3 = 143 g C

m2 WS7 = 146 g Cm2 Kochenderfer 2006) Finally we used pub-

lished values of AM root exudation rates on a per g root basis (Yin

Wheeler amp Phillips 2014) to estimate root C transfers assuming

that these inputs occurred only during the growing season Using

these model inputs we then ran two scenarios (1) the fertilized and

control watersheds had similar root C exudation rates per g of root

and (2) N fertilization reduced exudations rates by 25 We chose

this reduction in exudation rates to be conservative as it represents

around half of the decline in AM colonization we observed in the

fertilized watershed All model simulations were run for 30 year to

achieve steady state

3 | RESULTS

31 | Fine root metrics

Fine root biomass and the extent of mycorrhizal symbiosis in the

bulk mineral soil layer were significantly lower in the N-fertilized

watershed (Table 1) Overall the bulk mineral soil in the N-fertilized

watershed had 247 less standing fine root biomass than the refer-

ence watershed This reduction in fine root biomass was accompa-

nied by decreases in fine root surface area volume and the amount

of root forks per g Additionally N fertilization reduced AM

CARRARA ET AL | 5

colonization of fine roots by 559 Given the inherent limitations of

measuring root production using minirhizotron tubes or ingrowth

cores we use fine root biomass and AM colonization as proxies for

plant C allocation to gain nutrients While root turnover may be

enhanced under N fertilization we base the assumption that fine

root biomass is synonymous with belowground C allocation to fine

roots on our observations that fine root biomass was consistently

lower in the fertilized than control watersheds over all three of the

sequentially cored soil sampling dates during the growing season In

addition AM fungi have been shown to act as conduits for photo-

synthate C to rhizosphere microbes and as such represent an impor-

tant belowground flux of C (Kaiser et al 2015) Thus reduced

investment in AM colonization may also lead to reduced transfers of

photosynthate C to the rhizosphere resulting in an overall decline in

belowground C allocation

By contrast in the OH standing fine root biomass was larger in

the N-fertilized watershed although root biomass in the OH horizon

is markedly lower than in the bulk mineral soil (Table 1) However

like the bulk mineral soil AM colonization of OH roots was signifi-

cantly higher in the reference watershed (728 colonized) than the

N-fertilized watershed (609 colonized)

32 | Extracellular enzyme activity

Nearly all hydrolytic enzyme activities were lower under elevated N

conditions across all three soil fractions BG activity was significantly

lower in the N-fertilized watershed in both the bulk mineral (52

decrease) and OH (17 decrease) soil fractions (p lt 05 Figure 1a)

N fertilization significantly lowered AP activity in the bulk (12) and

rhizosphere (36) fractions of the mineral soil (p lt 05 Figure 1b)

Similarly N amendment significantly lowered NAG activity in both

bulk and rhizosphere fractions of the mineral soil by 41 and 37

respectively (p lt 05 Figure 1c) However BG activity in the rhizo-

sphere of the mineral soil was not significantly different between

0

04

08

12

16

Bulk Rhizo

AP

(m

olmiddot[

gdr

yso

il]ndash1

middot hr

ndash1)

0

1

2

3

4

5

OH0

01

02

03

04

Bulk Rhizo

BG

(m

olmiddot[g

dry

soi

l]ndash1

middot hr

ndash1)

0

05

1

15

2

OH

0

003

006

009

012

Bulk Rhizo

NA

G (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)

0

02

04

06

08

1

OH

0

04

08

12

16

Bulk Rhizo0

10

20

30

40

50

OH

0

025

05

075

1

Bulk Rhizo0

1

2

3

OH

0

1

2

3

4

5

Bulk Rhizo

Pero

xida

se (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)

0

1

2

3

4

OH

(a) (b)

(c) (d)

(e) (f)

Phen

ol o

xida

se (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)Pr

oteo

lysi

s (

gA

A-N

middot[g

dry

soil]

ndash1 middot

hrndash1

)

Reference

+N

F IGURE 1 N fertilization reduceshydrolytic and oxidative enzyme activitiesValues are the overall seasonal meanenzyme activities (mean SE) of (a) BG(b) AP (c) NAG (d) phenol oxidase (e)peroxidase and (f) proteolysis for each soilfraction (ie bulk rhizosphere and organichorizon) measured in June July andAugust 2015 across all plots (n = 10 plotsper watershed) Asterisks indicatesignificant differences in enzyme activitybetween watersheds within soil fractions(p lt 05) Note difference in scale betweenOH vs rhizosphere and bulk soil fractionsAA-N is amino acid nitrogen

6 | CARRARA ET AL

watersheds In addition AP and NAG activities in the OH horizon

did not vary by treatment

Liginolytic oxidative enzyme activities were also generally lower

in the N-fertilized watershed Phenol oxidase activity was signifi-

cantly lower in bulk (45 decrease) rhizosphere (49 decrease) and

OH horizons (57 decrease) of the fertilized watershed relative to

the reference watershed (p lt 05 Figure 1d) Peroxidase activity was

lower in the fertilized watershed in the bulk rhizosphere and OH

soil fractions with reductions of 30 25 and 36 respectively

(p lt 05 Figure 1e) Proteolytic enzyme activity was consistently

lower in each soil fraction under N fertilization with a 48 decrease

in bulk 56 decrease in rhizosphere and 40 decrease in OH

proteolysis (Figure 1f)

33 | Microbial community composition

When fungal taxonomic units were aggregated to the phylum level

there were no significant changes in the relative abundance of the

four most common fungal phyla or the unclassified group in any soil

horizon (Figure S1) When bacterial taxonomic units were aggregated

to the phylum level there were limited shifts in the relative abun-

dance of the seven most common bacterial phyla Relative abun-

dance of Actinobacteria was higher in the OH soil of the fertilized

watershed (Figure S2a) Relative abundance of Proteobacteria was

lower in the fertilized bulk soil and relative abundance of Firmicutes

was higher in both rhizosphere and bulk soil in the fertilized water-

shed (Figure S2bc)

Adonis analysis of bacterial communities revealed significant

effects for watershed soil horizon (p lt 001) and their interaction

(p = 02 total model R2 = 32) Within watersheds post hoc compar-

isons showed OH communities were different than bulk and rhizo-

sphere communities (Figure S3ab) There was no difference

between bulk and rhizosphere communities (Figure S3ab) Across

watersheds bacterial community composition differed in all soil frac-

tions such that OH rhizosphere and bulk soil exhibited unique com-

munities in the N-fertilized watershed compared to the reference

(Figure 2ace)

Adonis analysis of fungal communities revealed significant effects

of watershed and soil horizon (p lt 001 total model R2 = 12) but

not their interaction Within watersheds post hoc comparisons of

fungal communities showed OH communities were different than

bulk and rhizosphere communities in both watersheds but there was

no difference between bulk and rhizosphere communities

(Figure S3cd) Across watersheds fungal community composition

within OH and bulk soil fractions was significantly different (p lt 01

Figure 2bd) However there was no significant difference in fungal

communities between watersheds in rhizosphere soil (Figure 2f)

Comparison of bacterial and enzymatic NMDS scores across all

soil horizons showed a positive relationship between bacterial com-

munities and enzyme profiles (R2 = 48 p lt 01 Figure S4a) Compar-

ison of fungal and enzymatic NMDS scores across all soil horizons and

both watersheds showed no significant relationship between fungal

communities and enzyme profiles (Figure S4b) Across watersheds

comparison of bacterial and enzymatic NMDS scores showed a nega-

tive relationship between bacterial communities and enzyme profiles

in the bulk soil (R2 = 36 p lt 05 Figure 3a) but no significant rela-

tionship in rhizosphere soil Comparison of fungal and enzymatic

NMDS scores across watersheds showed no significant relationships

in either bulk or rhizosphere soil (Figure 3b) Linear regression of

AM colonization and the first bacterial NMDS axis resulted in a posi-

tive linear relationship in bulk soil (R2 = 063 p lt 001 Figure 3c) but

not rhizosphere soil There were no significant linear relationships

between AM colonization and the first fungal NMDS axis in either

soil fraction (Figure 3d)

34 | Net N mineralization and nitrification

The only significant difference we found in rates of nitrogen cycling

was that N mineralization and net nitrification in the OH were 40

and 51 higher in the fertilized watershed than the reference N

transformation rates did not significantly differ between watersheds

in either the bulk or rhizosphere soil fractions (Figure 4ab)

35 | Modeling potential impacts of plantndashmicrobiallinkages on soil decomposition

When we ran the model to steady state we found that reductions

in root biomass and root C transfers to the rhizosphere in the N-fer-

tilized watershed reduced microbial enzyme pools by ~16 and

enhanced soil C by ~3 compared to the reference watershed

(Table 2) Even though exudation rates on a per g root basis were

the same the fertilized watershed had lower overall exudations rates

at the ecosystem scale than the control watershed because of its

lower root biomass (Table 2) When we reduced exudation rates by

25 on a per g root basis in the fertilized watershed there was a

further exacerbation in the reduction in microbial enzyme activity to

a ~28 decline and a larger increase in soil C by ~20 (Table 2)

4 | DISCUSSION

Here we provide evidence that coupled interactions among plants

fungi and bacteria play an important role in enzyme activity

responses to N fertilization For fungi we observed distinct shifts in

fungal community structure in response to N fertilization (Figure 2b

d) but we found no evidence for a link between these shifts and

extracellular enzyme activity (Figure 3b) By contrast we found that

bacterial community composition shifts under N fertilization are cor-

related with declines in enzyme activity (Figure 3a) and that these

compositional shifts are tightly coupled to reductions in plant C allo-

cation to roots and mycorrhizal fungi (Figure 3c) Overall these

results suggest that N fertilization drives an integrated ecosystem

response whereby reductions in plant C allocation to roots and AM

fungi feedback on bacterial community structure and function

While whole-watershed fertilization at the Fernow results in a

pseudoreplicated experimental design (Hurlbert 1984) we conclude

CARRARA ET AL | 7

that the effects we measured are driven by N fertilization rather

than pre-existing differences between these adjacent watersheds

for four main reasons First soil chemistry (ie soil pH cation

exchange capacity nutrient content etc) were similar at the begin-

ning of the experiment (Adams amp Angradi 1996) Second the

amount of N added to the watershed yearly was originally chosen in

1989 to approximately double ambient N deposition rates but is

now more than quadruple current rates and as such it seems unli-

kely that this does not incur a biogeochemical response Third the

results from this watershed study are consistent with measurements

we made during the same year in a replicated N fertilization study

lt2 km away from these watersheds (the Fork Mountain Long-Term

Soil Productivity Plots Adams Burger Zelazny amp Baumgras 2004)

In this small-scale replicated study N fertilization reduced fine root

biomass and ligninolytic enzyme activity (Figure S5) Finally this

work builds upon other research at the Fernow that has found the

N-fertilized watershed had lower rates of litter decomposition

(Adams amp Angradi 1996) reduced understory richness (Gilliam et al

1994 2016 Walter Adams Gilliam amp Peterjohn 2017) and altered

N cycling (Adams et al 1993 Burnham Cumming Adams amp Peter-

john 2017 Gilliam Yurish amp Adams 2001 Gilliam et al 1996

2016) compared to the reference watershed

While we did observe significant declines in enzyme activity

across all three soil fractions particularly for the liginolytic enzymes

(Figure 1andashf) these declines were not correlated with significant

shifts in fungal community composition (Figure 3b) The lack of a

clear link between enzyme declines and changes in the fungal com-

munity does not support the prevailing paradigm that white-rot

Basidiomycota are the dominant cause of a decline in enzyme activi-

ties following N additions (Edwards Zak Kellner Eisenlord amp Pregit-

zer 2011 Fog 1988 Freedman Romanowicz Upchurch amp Zak

2015 Morrison et al 2016) It is possible that fungal enzyme activ-

ity response to N fertilization is independent of community composi-

tion (ie investment in enzyme activity declines with no change in

community structure) however microbial community composition

has been linked to catabolic functioning and enzyme activities across

N gradients and seasons (Fierer et al 2011 Vorıskova Brabcova

Cajthaml amp Baldrian 2014) Furthermore our data indicate that

ndash008

0

008

016

024

032

ndash09 ndash06 ndash03 0 03 06

Rhi

zo f

ungi

NM

DS2

Rhizo fungi NMDS1

ndash04

ndash02

0

02

04

ndash02 ndash01 0 01 02 03 04

Rhi

zo b

acte

ria

NM

DS2

Rhizo bacteria NMDS1

ndash02

ndash01

0

01

02

03

ndash02 0 02 04 06

Bul

k ba

cter

ia N

MD

S2

Bulk bacteria NMDS1

ndash04

ndash032

ndash024

ndash016

ndash008

0

ndash04 ndash02 0 02 04

OH

fun

gi N

MD

S2

OH fungi NMDS1

ndash06

ndash04

ndash02

0

02

ndash05 ndash04 ndash03 ndash02 ndash01 0

OH

bac

teri

a N

MD

S2

OH bacteria NMDS1

Reference

+N

ndash02

ndash01

0

01

02

03

04

ndash06 ndash04 ndash02 0 02 04

Bul

k fu

ngi N

MD

S2

Bulk fungi NMDS1

(a) (b)

(c) (d)

(e) (f)

plt01

plt01

plt01

plt01

plt01

F IGURE 2 N fertilization alteredbacterial community composition in OH (a)bulk (c) and rhizosphere (e) soils andfungal community composition in OH (b)and bulk (d) soils but not rhizosphere (f)All community data were obtained for eachsoil fraction in July 2015 (n = 10 plots perwatershed) p values indicate significantdifferences between the N-fertilized andreference community

8 | CARRARA ET AL

shifts in bacterial community composition under elevated N are cor-

related with reduced enzyme activity at the Fernow (Figure 3a)

While changes in bacterial community may be influenced by overall

fungal community composition these shifts in bacterial community

composition are tightly coupled to AM colonization (a metric of

belowground C allocation) suggesting that plant responses to N fer-

tilization feedback on bacterial community structure and function

(Figure 3c) While we cannot rule out that N-induced declines in

total fungal biomass led to reductions in liginolytic enzyme activity

(DeForest et al 2004 Frey et al 2004 Treseder 2008 Wallenstein

et al 2006) the dominance of AM trees in our plots whose inor-

ganic nutrient economy is largely driven by bacteria suggests that

free-living fungi may not be an important driver of N deposition

responses in AM-dominated systems (Cheeke et al 2017 Phillips

Brzostek amp Midgley 2013)

In contrast it appears that the declines in enzyme activity we

observed appear to be the result of a cascade of ecosystem

responses affecting both microbial community composition and plant

C allocation belowground The N-induced declines in fine root bio-

mass AM colonization and root morphology all indicate that there

was a reduction in the investment of C belowground by trees to gain

nutrients (Table 1) While previous research has shown that below-

ground C allocation is inversely correlated with N availability (Bae

Fahey Yanai amp Fisk 2015 Haynes amp Gower 1995 Phillips amp Fahey

2005) the tight linkage between the first bacterial NMDS axis and

AM colonization suggest that N-induced declines in belowground

C allocation caused a shift in the bacterial community composition

and subsequent reductions in enzyme activity (Figure 3ac) This

result also supports the emerging view that roots are active engi-

neers of microbial activity (Brzostek et al 2013 Finzi et al 2015

Yin et al 2014) and highlights the need to integrate root-microbial

interactions into our conceptual and predictive modeling frameworks

of how forest ecosystems respond to global change (Johnson

Gehring amp Jansa 2016 Terrer Vicca Hungate Phillips amp Prentice

2016)

An additional impact of the N fertilization treatment is that it

reduced soil pH (as measured in a 001 M calcium chloride buffer) of

the upper 5 cm of mineral soil in the fertilized watershed to 34

compared to 38 in the control watershed (Peterjohn unpublished

data) Soil pH is an important control on microbial community diver-

sity (Fierer et al 2012 Kaiser et al 2016 Lauber Hamady Knight

amp Fierer 2009) microbial biomass and enzyme activities (Rousk amp

Baath 2011 Sinsabaugh 2010) Thus the decline in soil pH may

account for a portion of the enzyme reductions we observed under

N fertilization To address this we assayed the sensitivity of phenol

oxidase and peroxidase enzyme activity in organic horizon bulk and

rhizosphere soils from the control watershed to three different levels

of pH that spanned a 1 unit shift (data not shown) Peroxidase activ-

ity was insensitive to pH (slope = 074 r2 = 05 p gt 05) While

phenol oxidase activity did significantly increase as pH increased

(slope = 010 r2 = 053 p lt 05) this sensitivity would only account

for a 10 decline in activity Given that we observed a 50 decline

in phenol oxidase in the treatment watershed coupled with the

greater overall importance of peroxidase enzymes in our study (ie

nearly an order of magnitude higher activity) it appears that pH is

an important but secondary driver of the enzyme activity responses

we observed

Over longer time scales the reductions in root and microbial

activity we observed at the Fernow may have important implications

for soil C stocks In our model simulation we found that feedbacks

between reductions in root biomass and enzyme production have

the potential to drive nearly a 3 increase in soil C stocks (Table 2)

When these were coupled with a 25 reduction in specific root C

exudation rates soil C in the fertilized watershed increased by nearly

20 over the 30-yr simulation This model was designed to be theo-

retical and as such it is used here to show the sensitivity of the

y = 0004x ndash 007R = 63

ndash02

0

02

04

06

0 25 50 75 100Bul

k B

acte

ria

OT

U N

MD

S1

AM Colonization ()

y = ndash0159x + 013R = 360

004

008

012

016

02

ndash02 0 02 04 06

Enz

yme

NM

DS

1

Bacteria OTU NMDS1

+NReference

0

004

008

012

016

02

ndash06 ndash04 ndash02 0 02 04

Enz

yme

NM

DS

1

Fungal OTU NMDS1

ndash06

ndash04

ndash02

0

02

04

0 25 50 75 100

Bul

k Fu

ngi

OT

U N

MD

S1

AM Colonization ()

plt05

plt001

(a)

(c)

(b)

(d)F IGURE 3 Bacterial but not fungalcommunity composition is correlated withthe first NMDS axis of enzyme activity (aamp b) Percent AM colonization is correlatedwith bacterial community composition (c)but not fungal community composition (d)Data presented are bulk soil communitycomposition data from July 2015 (n = 10plots per watershed) p values indicatesignificance of correlation

CARRARA ET AL | 9

system to the perturbations in plant inputs and is not meant to be

quantitatively predictive However this simple modeling effort pro-

vides compelling evidence that N-induced shifts in rootndashmicrobial

interactions have the potential to alter not only microbial production

of soil enzymes but also the size of the soil C pool To generate the

necessary data for fully integrating root-microbial interactions into

more sophisticated ecosystem models future research should couple

direct measurements of belowground C allocation responses to N

fertilization (eg total belowground C allocation root exudation)

with the resulting impacts on microbial activity

Given the prevailing paradigm that free-living fungi drive soil

enzyme activity and subsequent decomposition responses to N fer-

tilization (Carreiro et al 2000 Fog 1988 Waldrop et al 2004 Zak

et al 2008) our results raise an important question of why shifts in

plant C allocation bacterial community structure and enzyme activi-

ties were tightly coupled at the Fernow The divergent results

between the Fernow and other long-term fertilization sites may be

the result of differences in ambient N deposition and tree species

composition The Fernow is in a region that has historically received

some of the highest levels of N deposition in the United States

(Driscoll et al 2001) which may have enhanced bacterial dominance

before treatment began in 1989 By contrast N fertilization experi-

ments that have linked reductions in soil C decomposition to fungal

community composition and activity tend to be located in areas with

lower historical N deposition rates (Edwards et al 2011 Freedman

et al 2015 Frey et al 2004 Morrison et al 2016 Wallenstein

et al 2006) Second the Fernow is predominantly dominated by

AM trees whereas most N fertilization experiments have tended to

occur in ecosystems dominated by ECM trees with ECM-dominated

sites comprising ~80 of the studies included in the Janssens et al

(2010) meta-analysis AM trees show greater growth enhancements

with N addition (Thomas et al 2010) are characterized by lower

fungal to bacterial ratios (Cheeke et al 2017) and are less depen-

dent on plantndashmicrobial interactions to access N than ECM trees

(Brzostek Dragoni Brown amp Phillips 2015) As such in AM-domi-

nated systems like the Fernow bacteria may outweigh fungi in con-

trolling soil decomposition responses to N fertilization Additionally

evidence that ECM trees rely more on roots and rhizosphere

microbes to access N than AM trees suggests that N fertilization

may lead to greater declines in belowground C allocation and

enzyme activity than observed at the Fernow (Brzostek et al 2015

Yin et al 2014) Moving forward the equal distribution of ECM and

AM trees across the temperate forest landscape highlights a critical

need to investigate ECM and AM stand responses to N fertilization

within the same ecosystem (Midgley amp Phillips 2014)

Although enzyme declines were observed across all three soil

fractions (Figure 1andashf) N cycling shifts were only observed in the

OH where rates were elevated by nearly 50 and 40 for mineral-

ization and nitrification respectively (Figure 4ab) OH and mineral

soils differ in key biogeochemical traits and this may contribute to

these divergent N cycling responses In temperate forests OH

0

05

1

15

Bulk Rhizo

Net

N m

iner

aliz

atio

n (

gN

middotgndash1

middotday

ndash1)

Control

+N

0

3

6

9

12

15

18

OH

0

05

1

15

Bulk Rhizo

Net

nitr

ific

atio

n ((

gN

middotgndash1

middotday

ndash1)

0

2

4

6

8

10

12

OH

(a)

(b)

Reference

F IGURE 4 N fertilization increases (a) N mineralization and (b)nitrification in the OH but not the mineral soil Values are theoverall watershed-level mean rates (mean SE) measured in JuneJuly and August 2015 for each soil fraction (n = 10 plots perwatershed) Asterisks indicate significant differences betweentreatments (p lt 05)

TABLE 2 At model steady state fertilization reduced microbialenzyme pools by ~16 and enhanced soil C by ~3

Watershed ExudationEnzyme pool(mgcm3)

Soil C pool(mgcm3)

Control No change 00136 18473

Fertilized No change 00114 19092

25 reduction 00097 22116

When root C exudation was reduced 25 on a per g root basis in the

fertilized watershed there was a further exacerbation in the reduction in

microbial enzyme activity to a ~28 decline and a larger increase in soil

C by ~20

10 | CARRARA ET AL

typically have rapid N cycling rates reflecting greater organic matter

content and root densities than mineral soils (Brzostek amp Finzi

2011) Shifts in the ratio of gross immobilization to gross mineraliza-

tion may also play a role Net N mineralization rates increased in the

OH despite N fertilization induced declines in proteolytic and chiti-

nolytic enzyme activity that produce N monomers for microbial

uptake While we do not have data on gross N cycling we hypothe-

size that declines in gross microbial N immobilization due to reduced

N demand may have outpaced declines in gross N mineralization in

the fertilized watershed Regardless of the exact mechanism it is

important to put the OH results into context on a mass per unit

area basis the OH is less important to total ecosystem N cycling

than the underlying mineral soil

Much of the research on soil N fertilization has focused on

aboveground responses such as litter input and quality or fungal

community shifts to explain widely observed reductions in enzyme

activities and subsequent soil decomposition (Edwards et al 2011

Frey et al 2004 Morrison et al 2016 Sinsabaugh et al 2008

Waldrop et al 2004 Wallenstein et al 2006) Our results provide

evidence that enzyme activity declines under long-term N fertiliza-

tion at the Fernow are driven by a cascade of ecosystem responses

whereby reductions in belowground plant C investment lead to

shifts in bacterial community structure and a decline in their ability

to degrade soil organic matter Thus there is a need to integrate

plantndashmicrobial interactions into our current conceptual and predic-

tive models of N deposition impacts on temperate forests in order

to forecast soil C responses to shifting N deposition regimes

ACKNOWLEDGEMENTS

We acknowledge Mary Beth Adams Tom Schuler and the US Forest

Service staff at the Fernow Experimental Forest for logistical assis-

tance and access to the experimental watersheds and the LTSP site

This work was also supported by the National Science Foundation

Graduate Research Fellowship to Joseph Carrara under Grant No

DGE-1102689 and by the Long-Term Research in Environmental

Biology (LTREB) program at the National Science Foundation (Grant

Nos DEB-0417678 and DEB-1019522) to William Peterjohn Colin

Averill was supported by the NOAA Climate and Global Change

Postdoctoral Fellowship Program administered by Cooperative Pro-

grams for the Advancement of Earth System Science (CPAESS)

University Corporation for Atmospheric Research (UCAR) Boulder

Colorado USA We acknowledge the WVU Genomics Core Facility

Morgantown WV for support provided to help make this publication

possible We also thank Leah Baldinger Brittany Carver Mark Burn-

ham Hannah Hedrick Jennifer Mangano and Catherine Sesa for

assistance in the field and in the laboratory

ORCID

Joseph E Carrara httporcidorg0000-0003-0597-1175

Colin Averill httporcidorg0000-0003-4035-7760

REFERENCES

Adams M B amp Angradi T R (1996) Decomposition and nutrient

dynamics of hardwood leaf litter in the Femow Whole-Watershed

Acidification Experiment Forest Ecology and Management 1127 61ndash

69 httpsdoiorg1010160378-1127(95)03695-4

Adams M B Burger J Zelazny L amp Baumgras J (2004) Descrip-

tion of the fork mountain long-term soil productivity study Site

characterization USDA Forest Service General Technical Report NE

43 323

Adams M B Edwards P J Wood F amp Kochenderfer J N (1993)

Artificial watershed acidification on the Fernow Experimental Forest

USA Journal of Hydrology 150 505ndash519 httpsdoiorg101016

0022-1694(93)90123-Q

Allison S D Wallenstein M D amp Bradford M A (2010) Soil-carbon

response to warming dependent on microbial physiology Nature Geo-

science 3 336ndash340 httpsdoiorg101038ngeo846

Bae K Fahey T J Yanai R D amp Fisk M (2015) Soil nitrogen avail-

ability affects belowground carbon allocation and soil respiration in

northern hardwood forests of new hampshire Ecosystems 18 1179ndash

1191 httpsdoiorg101007s10021-015-9892-7

Brzostek E R Dragoni D Brown Z A amp Phillips R P (2015) Mycor-

rhizal type determines the magnitude and direction of root-induced

changes in decomposition in a temperate forest New Phytologist

206 1274ndash1282 httpsdoiorg101111nph13303

Brzostek E R amp Finzi A C (2011) Substrate supply fine roots and

temperature control proteolytic enzyme activity in temperate forest

soils Ecology 92 892ndash902 httpsdoiorg10189010-18031

Brzostek E R Fisher J B amp Phillips R P (2014) Modeling the carbon

cost of plant nitrogen acquisition Mycorrhizal trade-offs and multi-

path resistance uptake improve predictions of retranslocation Journal

of Geophysical Research Biogeosciences 119 1684ndash1697

Brzostek E R Greco A Drake J E amp Finzi A C (2013) Root carbon

inputs to the rhizosphere stimulate extracellular enzyme activity and

increase nitrogen availability in temperate forest soils Biogeochem-

istry 115 65ndash76 httpsdoiorg101007s10533-012-9818-9

Burnham M B Cumming J R Adams M B amp Peterjohn W T

(2017) Soluble soil aluminum alters the relative uptake of mineral

nitrogen forms by six mature temperate broadleaf tree species

possible implications for watershed nitrate retention Oecologia

185 1ndash11

Caporaso J G Kuczynski J Stombaugh J Bittinger K Bushman F

D Costello E K Huttley G A (2010) QIIME allows analysis of

high-throughput community sequencing data Nature Methods 7

335ndash336 httpsdoiorg101038nmethf303

Carreiro M M Sinsabaugh R L Repert D A amp Parkhurst D F

(2000) Microbial enzyme shifts explain litter decay responses to sim-

ulated nitrogen deposition Ecology 81 2359ndash2365 httpsdoiorg

1018900012-9658(2000)081[2359MESELD]20CO2

Cheeke T E Phillips R P Brzostek E R Rosling A Bever J D amp

Fransson P (2017) Dominant mycorrhizal association of trees alters

carbon and nutrient cycling by selecting for microbial groups with

distinct enzyme function New Phytologist 214 432ndash442 httpsdoi

org101111nph14343

Cheng W Parton W J Gonzalez-Meler M A Phillips R Asao S

McNickle G G Jastrow J D (2014) Synthesis and modeling

perspectives of rhizosphere priming New Phytologist 201 31ndash44

httpsdoiorg101111nph12440

Comas L H Callahan H S amp Midford P E (2014) Patterns in root

traits of woody species hosting arbuscular and ectomycorrhizas

Implications for the evolution of belowground strategies Ecology and

Evolution 4 2979ndash2990 httpsdoiorg101002ece31147

Datta R Kelkar A Baraniya D Molaei A Moulick A Meena R S amp

Formanek P (2017) Enzymatic degradation of lignin in soil A

review Sustainability 9 1163 httpsdoiorg103390su9071163

CARRARA ET AL | 11

reference databases To determine the effect of N fertilization on

overall species composition the top four abundant fungal phyla

(Ascomycota Basidiomycota Chytridiomycota and Zygotmycota as

well as lsquounclassifiedrsquo) were examined for shifts in relative abundance

To further analyze fungal and bacterial community composition

each OTU table was proportionally normalized such that the per OTU

sequence count within a sample was divided by the total number of

sequences detected in a given sample generating a proportion on the

interval [01] This proportional normalization has been demonstrated

to be sufficiently accurate when using the BrayndashCurtis similarity met-

ric (Weiss et al 2017) We then used normalized OTU tables to calcu-

late BrayndashCurtis similarity using the vegdist function within the vegan

package for R statistical software (Oksanen et al 2015 R Core Team

2017) Community similarity was analyzed as a function of watershed

soil horizon and their interaction using the adonis function within the

vegan package If interactions were not significant then they were

removed and we only analyzed main effects

To understand how microbial community composition related to

variation in soil enzyme profiles we created similarity matrices of

soil enzymes We calculated BrayndashCurtis similarity of enzyme profiles

using all enzymes using the vegdist function in vegan We then per-

formed nonmetric multidimensional scaling (NMDS) to generate

NMDS scores for both microbial and enzymatic profiles We tested

for relationships between microbial communities and enzymatic pro-

files in bulk and rhizosphere soil separately by comparing the first

NMDS axes of microbial and enzymatic profiles using linear regres-

sion in SAS JMP vs 1002 (SAS Institute Cary NC USA) We fur-

ther examined the relationship between microbial communities and

enzymatic profiles across all soil horizons by comparing the first

NMDS axes of microbial and enzymatic profiles using linear regres-

sion across both watersheds

To assess the relative importance of belowground C allocation

(ie AM colonization) on fungal and bacterial community composi-

tion we explored whether there were correlations between AM

colonization and the first NMDS axis of bacterial and fungal commu-

nity composition in bulk and rhizosphere soil separately using SAS

JMP vs 1002 (SAS Institute Cary NC USA)

26 | Net mineralization and nitrification

For all soil fractions at each sampling date we measured the rate of

net N mineralization and net nitrification within 48 hours of collec-

tion Net N mineralization and nitrification rates were expressed as

the difference in the 1 M KCl extractable pool sizes of NHthorn4 and

NO3 between an initial sample and a sample that was aerobically

incubated in the lab for 14 days at room temperature and under

constant soil moisture (Finzi Van Breemen amp Canham 1998) To

perform the extractions 50 ml of 1 M KCl was added to a jar con-

taining 5 g of each soil sample Samples were sealed and shaken for

30 min and allowed to settle for 2 hr Samples were filtered using

Pall Supor 450 filters via syringe into 20-ml vials and stored at

20degC prior to analysis by flow injection (Lachat QuikChem 8500

series 2)

27 | Statistical analysis

To determine the extent to which N fertilization altered enzyme

activities N mineralization nitrification and root metrics we per-

formed a three-way analysis of variance (ANOVA) in SAS JMP vs

1002 (SAS Institute Cary NC USA) and used date watershed and

soil horizon as factors Although we included date in our statistical

models we present growing season means in the results reflecting

our focus on treatment rather than seasonal effects Post hoc multi-

ple comparisons were also made among watersheds dates and soil

fractions using the TukeyndashKramer HSD test

28 | Modeling potential impacts of plantndashmicrobiallinkages on soil decomposition

To further examine the extent to which linkages between plants and

soil microbes drive soil enzyme responses to N fertilization as well

as to estimate potential impacts on soil C stocks we used a micro-

bial enzyme decomposition model (Allison Wallenstein amp Bradford

2010) that was modified in Cheng et al (2014) to include seasonal

differences in soil temperature the inputs of root and leaf litter and

root transfers of C to the rhizosphere For leaf and root litter inputs

we used the mean observed litterfall rate from 1998 to 2015 for

each watershed (Kochenderfer 2006 Peterjohn et al 2017) and our

own measurements of root biomass with the assumption that roots

in both watersheds turnover once each year (McCormack Adams

Smithwick amp Eissenstat 2017) For both roots and leaves we

assumed that turnover primarily occurred during the leaf senescence

in the fall It is important to note there are no significant differences

in leaf litterfall rates between the two watersheds (WS3 = 143 g C

m2 WS7 = 146 g Cm2 Kochenderfer 2006) Finally we used pub-

lished values of AM root exudation rates on a per g root basis (Yin

Wheeler amp Phillips 2014) to estimate root C transfers assuming

that these inputs occurred only during the growing season Using

these model inputs we then ran two scenarios (1) the fertilized and

control watersheds had similar root C exudation rates per g of root

and (2) N fertilization reduced exudations rates by 25 We chose

this reduction in exudation rates to be conservative as it represents

around half of the decline in AM colonization we observed in the

fertilized watershed All model simulations were run for 30 year to

achieve steady state

3 | RESULTS

31 | Fine root metrics

Fine root biomass and the extent of mycorrhizal symbiosis in the

bulk mineral soil layer were significantly lower in the N-fertilized

watershed (Table 1) Overall the bulk mineral soil in the N-fertilized

watershed had 247 less standing fine root biomass than the refer-

ence watershed This reduction in fine root biomass was accompa-

nied by decreases in fine root surface area volume and the amount

of root forks per g Additionally N fertilization reduced AM

CARRARA ET AL | 5

colonization of fine roots by 559 Given the inherent limitations of

measuring root production using minirhizotron tubes or ingrowth

cores we use fine root biomass and AM colonization as proxies for

plant C allocation to gain nutrients While root turnover may be

enhanced under N fertilization we base the assumption that fine

root biomass is synonymous with belowground C allocation to fine

roots on our observations that fine root biomass was consistently

lower in the fertilized than control watersheds over all three of the

sequentially cored soil sampling dates during the growing season In

addition AM fungi have been shown to act as conduits for photo-

synthate C to rhizosphere microbes and as such represent an impor-

tant belowground flux of C (Kaiser et al 2015) Thus reduced

investment in AM colonization may also lead to reduced transfers of

photosynthate C to the rhizosphere resulting in an overall decline in

belowground C allocation

By contrast in the OH standing fine root biomass was larger in

the N-fertilized watershed although root biomass in the OH horizon

is markedly lower than in the bulk mineral soil (Table 1) However

like the bulk mineral soil AM colonization of OH roots was signifi-

cantly higher in the reference watershed (728 colonized) than the

N-fertilized watershed (609 colonized)

32 | Extracellular enzyme activity

Nearly all hydrolytic enzyme activities were lower under elevated N

conditions across all three soil fractions BG activity was significantly

lower in the N-fertilized watershed in both the bulk mineral (52

decrease) and OH (17 decrease) soil fractions (p lt 05 Figure 1a)

N fertilization significantly lowered AP activity in the bulk (12) and

rhizosphere (36) fractions of the mineral soil (p lt 05 Figure 1b)

Similarly N amendment significantly lowered NAG activity in both

bulk and rhizosphere fractions of the mineral soil by 41 and 37

respectively (p lt 05 Figure 1c) However BG activity in the rhizo-

sphere of the mineral soil was not significantly different between

0

04

08

12

16

Bulk Rhizo

AP

(m

olmiddot[

gdr

yso

il]ndash1

middot hr

ndash1)

0

1

2

3

4

5

OH0

01

02

03

04

Bulk Rhizo

BG

(m

olmiddot[g

dry

soi

l]ndash1

middot hr

ndash1)

0

05

1

15

2

OH

0

003

006

009

012

Bulk Rhizo

NA

G (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)

0

02

04

06

08

1

OH

0

04

08

12

16

Bulk Rhizo0

10

20

30

40

50

OH

0

025

05

075

1

Bulk Rhizo0

1

2

3

OH

0

1

2

3

4

5

Bulk Rhizo

Pero

xida

se (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)

0

1

2

3

4

OH

(a) (b)

(c) (d)

(e) (f)

Phen

ol o

xida

se (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)Pr

oteo

lysi

s (

gA

A-N

middot[g

dry

soil]

ndash1 middot

hrndash1

)

Reference

+N

F IGURE 1 N fertilization reduceshydrolytic and oxidative enzyme activitiesValues are the overall seasonal meanenzyme activities (mean SE) of (a) BG(b) AP (c) NAG (d) phenol oxidase (e)peroxidase and (f) proteolysis for each soilfraction (ie bulk rhizosphere and organichorizon) measured in June July andAugust 2015 across all plots (n = 10 plotsper watershed) Asterisks indicatesignificant differences in enzyme activitybetween watersheds within soil fractions(p lt 05) Note difference in scale betweenOH vs rhizosphere and bulk soil fractionsAA-N is amino acid nitrogen

6 | CARRARA ET AL

watersheds In addition AP and NAG activities in the OH horizon

did not vary by treatment

Liginolytic oxidative enzyme activities were also generally lower

in the N-fertilized watershed Phenol oxidase activity was signifi-

cantly lower in bulk (45 decrease) rhizosphere (49 decrease) and

OH horizons (57 decrease) of the fertilized watershed relative to

the reference watershed (p lt 05 Figure 1d) Peroxidase activity was

lower in the fertilized watershed in the bulk rhizosphere and OH

soil fractions with reductions of 30 25 and 36 respectively

(p lt 05 Figure 1e) Proteolytic enzyme activity was consistently

lower in each soil fraction under N fertilization with a 48 decrease

in bulk 56 decrease in rhizosphere and 40 decrease in OH

proteolysis (Figure 1f)

33 | Microbial community composition

When fungal taxonomic units were aggregated to the phylum level

there were no significant changes in the relative abundance of the

four most common fungal phyla or the unclassified group in any soil

horizon (Figure S1) When bacterial taxonomic units were aggregated

to the phylum level there were limited shifts in the relative abun-

dance of the seven most common bacterial phyla Relative abun-

dance of Actinobacteria was higher in the OH soil of the fertilized

watershed (Figure S2a) Relative abundance of Proteobacteria was

lower in the fertilized bulk soil and relative abundance of Firmicutes

was higher in both rhizosphere and bulk soil in the fertilized water-

shed (Figure S2bc)

Adonis analysis of bacterial communities revealed significant

effects for watershed soil horizon (p lt 001) and their interaction

(p = 02 total model R2 = 32) Within watersheds post hoc compar-

isons showed OH communities were different than bulk and rhizo-

sphere communities (Figure S3ab) There was no difference

between bulk and rhizosphere communities (Figure S3ab) Across

watersheds bacterial community composition differed in all soil frac-

tions such that OH rhizosphere and bulk soil exhibited unique com-

munities in the N-fertilized watershed compared to the reference

(Figure 2ace)

Adonis analysis of fungal communities revealed significant effects

of watershed and soil horizon (p lt 001 total model R2 = 12) but

not their interaction Within watersheds post hoc comparisons of

fungal communities showed OH communities were different than

bulk and rhizosphere communities in both watersheds but there was

no difference between bulk and rhizosphere communities

(Figure S3cd) Across watersheds fungal community composition

within OH and bulk soil fractions was significantly different (p lt 01

Figure 2bd) However there was no significant difference in fungal

communities between watersheds in rhizosphere soil (Figure 2f)

Comparison of bacterial and enzymatic NMDS scores across all

soil horizons showed a positive relationship between bacterial com-

munities and enzyme profiles (R2 = 48 p lt 01 Figure S4a) Compar-

ison of fungal and enzymatic NMDS scores across all soil horizons and

both watersheds showed no significant relationship between fungal

communities and enzyme profiles (Figure S4b) Across watersheds

comparison of bacterial and enzymatic NMDS scores showed a nega-

tive relationship between bacterial communities and enzyme profiles

in the bulk soil (R2 = 36 p lt 05 Figure 3a) but no significant rela-

tionship in rhizosphere soil Comparison of fungal and enzymatic

NMDS scores across watersheds showed no significant relationships

in either bulk or rhizosphere soil (Figure 3b) Linear regression of

AM colonization and the first bacterial NMDS axis resulted in a posi-

tive linear relationship in bulk soil (R2 = 063 p lt 001 Figure 3c) but

not rhizosphere soil There were no significant linear relationships

between AM colonization and the first fungal NMDS axis in either

soil fraction (Figure 3d)

34 | Net N mineralization and nitrification

The only significant difference we found in rates of nitrogen cycling

was that N mineralization and net nitrification in the OH were 40

and 51 higher in the fertilized watershed than the reference N

transformation rates did not significantly differ between watersheds

in either the bulk or rhizosphere soil fractions (Figure 4ab)

35 | Modeling potential impacts of plantndashmicrobiallinkages on soil decomposition

When we ran the model to steady state we found that reductions

in root biomass and root C transfers to the rhizosphere in the N-fer-

tilized watershed reduced microbial enzyme pools by ~16 and

enhanced soil C by ~3 compared to the reference watershed

(Table 2) Even though exudation rates on a per g root basis were

the same the fertilized watershed had lower overall exudations rates

at the ecosystem scale than the control watershed because of its

lower root biomass (Table 2) When we reduced exudation rates by

25 on a per g root basis in the fertilized watershed there was a

further exacerbation in the reduction in microbial enzyme activity to

a ~28 decline and a larger increase in soil C by ~20 (Table 2)

4 | DISCUSSION

Here we provide evidence that coupled interactions among plants

fungi and bacteria play an important role in enzyme activity

responses to N fertilization For fungi we observed distinct shifts in

fungal community structure in response to N fertilization (Figure 2b

d) but we found no evidence for a link between these shifts and

extracellular enzyme activity (Figure 3b) By contrast we found that

bacterial community composition shifts under N fertilization are cor-

related with declines in enzyme activity (Figure 3a) and that these

compositional shifts are tightly coupled to reductions in plant C allo-

cation to roots and mycorrhizal fungi (Figure 3c) Overall these

results suggest that N fertilization drives an integrated ecosystem

response whereby reductions in plant C allocation to roots and AM

fungi feedback on bacterial community structure and function

While whole-watershed fertilization at the Fernow results in a

pseudoreplicated experimental design (Hurlbert 1984) we conclude

CARRARA ET AL | 7

that the effects we measured are driven by N fertilization rather

than pre-existing differences between these adjacent watersheds

for four main reasons First soil chemistry (ie soil pH cation

exchange capacity nutrient content etc) were similar at the begin-

ning of the experiment (Adams amp Angradi 1996) Second the

amount of N added to the watershed yearly was originally chosen in

1989 to approximately double ambient N deposition rates but is

now more than quadruple current rates and as such it seems unli-

kely that this does not incur a biogeochemical response Third the

results from this watershed study are consistent with measurements

we made during the same year in a replicated N fertilization study

lt2 km away from these watersheds (the Fork Mountain Long-Term

Soil Productivity Plots Adams Burger Zelazny amp Baumgras 2004)

In this small-scale replicated study N fertilization reduced fine root

biomass and ligninolytic enzyme activity (Figure S5) Finally this

work builds upon other research at the Fernow that has found the

N-fertilized watershed had lower rates of litter decomposition

(Adams amp Angradi 1996) reduced understory richness (Gilliam et al

1994 2016 Walter Adams Gilliam amp Peterjohn 2017) and altered

N cycling (Adams et al 1993 Burnham Cumming Adams amp Peter-

john 2017 Gilliam Yurish amp Adams 2001 Gilliam et al 1996

2016) compared to the reference watershed

While we did observe significant declines in enzyme activity

across all three soil fractions particularly for the liginolytic enzymes

(Figure 1andashf) these declines were not correlated with significant

shifts in fungal community composition (Figure 3b) The lack of a

clear link between enzyme declines and changes in the fungal com-

munity does not support the prevailing paradigm that white-rot

Basidiomycota are the dominant cause of a decline in enzyme activi-

ties following N additions (Edwards Zak Kellner Eisenlord amp Pregit-

zer 2011 Fog 1988 Freedman Romanowicz Upchurch amp Zak

2015 Morrison et al 2016) It is possible that fungal enzyme activ-

ity response to N fertilization is independent of community composi-

tion (ie investment in enzyme activity declines with no change in

community structure) however microbial community composition

has been linked to catabolic functioning and enzyme activities across

N gradients and seasons (Fierer et al 2011 Vorıskova Brabcova

Cajthaml amp Baldrian 2014) Furthermore our data indicate that

ndash008

0

008

016

024

032

ndash09 ndash06 ndash03 0 03 06

Rhi

zo f

ungi

NM

DS2

Rhizo fungi NMDS1

ndash04

ndash02

0

02

04

ndash02 ndash01 0 01 02 03 04

Rhi

zo b

acte

ria

NM

DS2

Rhizo bacteria NMDS1

ndash02

ndash01

0

01

02

03

ndash02 0 02 04 06

Bul

k ba

cter

ia N

MD

S2

Bulk bacteria NMDS1

ndash04

ndash032

ndash024

ndash016

ndash008

0

ndash04 ndash02 0 02 04

OH

fun

gi N

MD

S2

OH fungi NMDS1

ndash06

ndash04

ndash02

0

02

ndash05 ndash04 ndash03 ndash02 ndash01 0

OH

bac

teri

a N

MD

S2

OH bacteria NMDS1

Reference

+N

ndash02

ndash01

0

01

02

03

04

ndash06 ndash04 ndash02 0 02 04

Bul

k fu

ngi N

MD

S2

Bulk fungi NMDS1

(a) (b)

(c) (d)

(e) (f)

plt01

plt01

plt01

plt01

plt01

F IGURE 2 N fertilization alteredbacterial community composition in OH (a)bulk (c) and rhizosphere (e) soils andfungal community composition in OH (b)and bulk (d) soils but not rhizosphere (f)All community data were obtained for eachsoil fraction in July 2015 (n = 10 plots perwatershed) p values indicate significantdifferences between the N-fertilized andreference community

8 | CARRARA ET AL

shifts in bacterial community composition under elevated N are cor-

related with reduced enzyme activity at the Fernow (Figure 3a)

While changes in bacterial community may be influenced by overall

fungal community composition these shifts in bacterial community

composition are tightly coupled to AM colonization (a metric of

belowground C allocation) suggesting that plant responses to N fer-

tilization feedback on bacterial community structure and function

(Figure 3c) While we cannot rule out that N-induced declines in

total fungal biomass led to reductions in liginolytic enzyme activity

(DeForest et al 2004 Frey et al 2004 Treseder 2008 Wallenstein

et al 2006) the dominance of AM trees in our plots whose inor-

ganic nutrient economy is largely driven by bacteria suggests that

free-living fungi may not be an important driver of N deposition

responses in AM-dominated systems (Cheeke et al 2017 Phillips

Brzostek amp Midgley 2013)

In contrast it appears that the declines in enzyme activity we

observed appear to be the result of a cascade of ecosystem

responses affecting both microbial community composition and plant

C allocation belowground The N-induced declines in fine root bio-

mass AM colonization and root morphology all indicate that there

was a reduction in the investment of C belowground by trees to gain

nutrients (Table 1) While previous research has shown that below-

ground C allocation is inversely correlated with N availability (Bae

Fahey Yanai amp Fisk 2015 Haynes amp Gower 1995 Phillips amp Fahey

2005) the tight linkage between the first bacterial NMDS axis and

AM colonization suggest that N-induced declines in belowground

C allocation caused a shift in the bacterial community composition

and subsequent reductions in enzyme activity (Figure 3ac) This

result also supports the emerging view that roots are active engi-

neers of microbial activity (Brzostek et al 2013 Finzi et al 2015

Yin et al 2014) and highlights the need to integrate root-microbial

interactions into our conceptual and predictive modeling frameworks

of how forest ecosystems respond to global change (Johnson

Gehring amp Jansa 2016 Terrer Vicca Hungate Phillips amp Prentice

2016)

An additional impact of the N fertilization treatment is that it

reduced soil pH (as measured in a 001 M calcium chloride buffer) of

the upper 5 cm of mineral soil in the fertilized watershed to 34

compared to 38 in the control watershed (Peterjohn unpublished

data) Soil pH is an important control on microbial community diver-

sity (Fierer et al 2012 Kaiser et al 2016 Lauber Hamady Knight

amp Fierer 2009) microbial biomass and enzyme activities (Rousk amp

Baath 2011 Sinsabaugh 2010) Thus the decline in soil pH may

account for a portion of the enzyme reductions we observed under

N fertilization To address this we assayed the sensitivity of phenol

oxidase and peroxidase enzyme activity in organic horizon bulk and

rhizosphere soils from the control watershed to three different levels

of pH that spanned a 1 unit shift (data not shown) Peroxidase activ-

ity was insensitive to pH (slope = 074 r2 = 05 p gt 05) While

phenol oxidase activity did significantly increase as pH increased

(slope = 010 r2 = 053 p lt 05) this sensitivity would only account

for a 10 decline in activity Given that we observed a 50 decline

in phenol oxidase in the treatment watershed coupled with the

greater overall importance of peroxidase enzymes in our study (ie

nearly an order of magnitude higher activity) it appears that pH is

an important but secondary driver of the enzyme activity responses

we observed

Over longer time scales the reductions in root and microbial

activity we observed at the Fernow may have important implications

for soil C stocks In our model simulation we found that feedbacks

between reductions in root biomass and enzyme production have

the potential to drive nearly a 3 increase in soil C stocks (Table 2)

When these were coupled with a 25 reduction in specific root C

exudation rates soil C in the fertilized watershed increased by nearly

20 over the 30-yr simulation This model was designed to be theo-

retical and as such it is used here to show the sensitivity of the

y = 0004x ndash 007R = 63

ndash02

0

02

04

06

0 25 50 75 100Bul

k B

acte

ria

OT

U N

MD

S1

AM Colonization ()

y = ndash0159x + 013R = 360

004

008

012

016

02

ndash02 0 02 04 06

Enz

yme

NM

DS

1

Bacteria OTU NMDS1

+NReference

0

004

008

012

016

02

ndash06 ndash04 ndash02 0 02 04

Enz

yme

NM

DS

1

Fungal OTU NMDS1

ndash06

ndash04

ndash02

0

02

04

0 25 50 75 100

Bul

k Fu

ngi

OT

U N

MD

S1

AM Colonization ()

plt05

plt001

(a)

(c)

(b)

(d)F IGURE 3 Bacterial but not fungalcommunity composition is correlated withthe first NMDS axis of enzyme activity (aamp b) Percent AM colonization is correlatedwith bacterial community composition (c)but not fungal community composition (d)Data presented are bulk soil communitycomposition data from July 2015 (n = 10plots per watershed) p values indicatesignificance of correlation

CARRARA ET AL | 9

system to the perturbations in plant inputs and is not meant to be

quantitatively predictive However this simple modeling effort pro-

vides compelling evidence that N-induced shifts in rootndashmicrobial

interactions have the potential to alter not only microbial production

of soil enzymes but also the size of the soil C pool To generate the

necessary data for fully integrating root-microbial interactions into

more sophisticated ecosystem models future research should couple

direct measurements of belowground C allocation responses to N

fertilization (eg total belowground C allocation root exudation)

with the resulting impacts on microbial activity

Given the prevailing paradigm that free-living fungi drive soil

enzyme activity and subsequent decomposition responses to N fer-

tilization (Carreiro et al 2000 Fog 1988 Waldrop et al 2004 Zak

et al 2008) our results raise an important question of why shifts in

plant C allocation bacterial community structure and enzyme activi-

ties were tightly coupled at the Fernow The divergent results

between the Fernow and other long-term fertilization sites may be

the result of differences in ambient N deposition and tree species

composition The Fernow is in a region that has historically received

some of the highest levels of N deposition in the United States

(Driscoll et al 2001) which may have enhanced bacterial dominance

before treatment began in 1989 By contrast N fertilization experi-

ments that have linked reductions in soil C decomposition to fungal

community composition and activity tend to be located in areas with

lower historical N deposition rates (Edwards et al 2011 Freedman

et al 2015 Frey et al 2004 Morrison et al 2016 Wallenstein

et al 2006) Second the Fernow is predominantly dominated by

AM trees whereas most N fertilization experiments have tended to

occur in ecosystems dominated by ECM trees with ECM-dominated

sites comprising ~80 of the studies included in the Janssens et al

(2010) meta-analysis AM trees show greater growth enhancements

with N addition (Thomas et al 2010) are characterized by lower

fungal to bacterial ratios (Cheeke et al 2017) and are less depen-

dent on plantndashmicrobial interactions to access N than ECM trees

(Brzostek Dragoni Brown amp Phillips 2015) As such in AM-domi-

nated systems like the Fernow bacteria may outweigh fungi in con-

trolling soil decomposition responses to N fertilization Additionally

evidence that ECM trees rely more on roots and rhizosphere

microbes to access N than AM trees suggests that N fertilization

may lead to greater declines in belowground C allocation and

enzyme activity than observed at the Fernow (Brzostek et al 2015

Yin et al 2014) Moving forward the equal distribution of ECM and

AM trees across the temperate forest landscape highlights a critical

need to investigate ECM and AM stand responses to N fertilization

within the same ecosystem (Midgley amp Phillips 2014)

Although enzyme declines were observed across all three soil

fractions (Figure 1andashf) N cycling shifts were only observed in the

OH where rates were elevated by nearly 50 and 40 for mineral-

ization and nitrification respectively (Figure 4ab) OH and mineral

soils differ in key biogeochemical traits and this may contribute to

these divergent N cycling responses In temperate forests OH

0

05

1

15

Bulk Rhizo

Net

N m

iner

aliz

atio

n (

gN

middotgndash1

middotday

ndash1)

Control

+N

0

3

6

9

12

15

18

OH

0

05

1

15

Bulk Rhizo

Net

nitr

ific

atio

n ((

gN

middotgndash1

middotday

ndash1)

0

2

4

6

8

10

12

OH

(a)

(b)

Reference

F IGURE 4 N fertilization increases (a) N mineralization and (b)nitrification in the OH but not the mineral soil Values are theoverall watershed-level mean rates (mean SE) measured in JuneJuly and August 2015 for each soil fraction (n = 10 plots perwatershed) Asterisks indicate significant differences betweentreatments (p lt 05)

TABLE 2 At model steady state fertilization reduced microbialenzyme pools by ~16 and enhanced soil C by ~3

Watershed ExudationEnzyme pool(mgcm3)

Soil C pool(mgcm3)

Control No change 00136 18473

Fertilized No change 00114 19092

25 reduction 00097 22116

When root C exudation was reduced 25 on a per g root basis in the

fertilized watershed there was a further exacerbation in the reduction in

microbial enzyme activity to a ~28 decline and a larger increase in soil

C by ~20

10 | CARRARA ET AL

typically have rapid N cycling rates reflecting greater organic matter

content and root densities than mineral soils (Brzostek amp Finzi

2011) Shifts in the ratio of gross immobilization to gross mineraliza-

tion may also play a role Net N mineralization rates increased in the

OH despite N fertilization induced declines in proteolytic and chiti-

nolytic enzyme activity that produce N monomers for microbial

uptake While we do not have data on gross N cycling we hypothe-

size that declines in gross microbial N immobilization due to reduced

N demand may have outpaced declines in gross N mineralization in

the fertilized watershed Regardless of the exact mechanism it is

important to put the OH results into context on a mass per unit

area basis the OH is less important to total ecosystem N cycling

than the underlying mineral soil

Much of the research on soil N fertilization has focused on

aboveground responses such as litter input and quality or fungal

community shifts to explain widely observed reductions in enzyme

activities and subsequent soil decomposition (Edwards et al 2011

Frey et al 2004 Morrison et al 2016 Sinsabaugh et al 2008

Waldrop et al 2004 Wallenstein et al 2006) Our results provide

evidence that enzyme activity declines under long-term N fertiliza-

tion at the Fernow are driven by a cascade of ecosystem responses

whereby reductions in belowground plant C investment lead to

shifts in bacterial community structure and a decline in their ability

to degrade soil organic matter Thus there is a need to integrate

plantndashmicrobial interactions into our current conceptual and predic-

tive models of N deposition impacts on temperate forests in order

to forecast soil C responses to shifting N deposition regimes

ACKNOWLEDGEMENTS

We acknowledge Mary Beth Adams Tom Schuler and the US Forest

Service staff at the Fernow Experimental Forest for logistical assis-

tance and access to the experimental watersheds and the LTSP site

This work was also supported by the National Science Foundation

Graduate Research Fellowship to Joseph Carrara under Grant No

DGE-1102689 and by the Long-Term Research in Environmental

Biology (LTREB) program at the National Science Foundation (Grant

Nos DEB-0417678 and DEB-1019522) to William Peterjohn Colin

Averill was supported by the NOAA Climate and Global Change

Postdoctoral Fellowship Program administered by Cooperative Pro-

grams for the Advancement of Earth System Science (CPAESS)

University Corporation for Atmospheric Research (UCAR) Boulder

Colorado USA We acknowledge the WVU Genomics Core Facility

Morgantown WV for support provided to help make this publication

possible We also thank Leah Baldinger Brittany Carver Mark Burn-

ham Hannah Hedrick Jennifer Mangano and Catherine Sesa for

assistance in the field and in the laboratory

ORCID

Joseph E Carrara httporcidorg0000-0003-0597-1175

Colin Averill httporcidorg0000-0003-4035-7760

REFERENCES

Adams M B amp Angradi T R (1996) Decomposition and nutrient

dynamics of hardwood leaf litter in the Femow Whole-Watershed

Acidification Experiment Forest Ecology and Management 1127 61ndash

69 httpsdoiorg1010160378-1127(95)03695-4

Adams M B Burger J Zelazny L amp Baumgras J (2004) Descrip-

tion of the fork mountain long-term soil productivity study Site

characterization USDA Forest Service General Technical Report NE

43 323

Adams M B Edwards P J Wood F amp Kochenderfer J N (1993)

Artificial watershed acidification on the Fernow Experimental Forest

USA Journal of Hydrology 150 505ndash519 httpsdoiorg101016

0022-1694(93)90123-Q

Allison S D Wallenstein M D amp Bradford M A (2010) Soil-carbon

response to warming dependent on microbial physiology Nature Geo-

science 3 336ndash340 httpsdoiorg101038ngeo846

Bae K Fahey T J Yanai R D amp Fisk M (2015) Soil nitrogen avail-

ability affects belowground carbon allocation and soil respiration in

northern hardwood forests of new hampshire Ecosystems 18 1179ndash

1191 httpsdoiorg101007s10021-015-9892-7

Brzostek E R Dragoni D Brown Z A amp Phillips R P (2015) Mycor-

rhizal type determines the magnitude and direction of root-induced

changes in decomposition in a temperate forest New Phytologist

206 1274ndash1282 httpsdoiorg101111nph13303

Brzostek E R amp Finzi A C (2011) Substrate supply fine roots and

temperature control proteolytic enzyme activity in temperate forest

soils Ecology 92 892ndash902 httpsdoiorg10189010-18031

Brzostek E R Fisher J B amp Phillips R P (2014) Modeling the carbon

cost of plant nitrogen acquisition Mycorrhizal trade-offs and multi-

path resistance uptake improve predictions of retranslocation Journal

of Geophysical Research Biogeosciences 119 1684ndash1697

Brzostek E R Greco A Drake J E amp Finzi A C (2013) Root carbon

inputs to the rhizosphere stimulate extracellular enzyme activity and

increase nitrogen availability in temperate forest soils Biogeochem-

istry 115 65ndash76 httpsdoiorg101007s10533-012-9818-9

Burnham M B Cumming J R Adams M B amp Peterjohn W T

(2017) Soluble soil aluminum alters the relative uptake of mineral

nitrogen forms by six mature temperate broadleaf tree species

possible implications for watershed nitrate retention Oecologia

185 1ndash11

Caporaso J G Kuczynski J Stombaugh J Bittinger K Bushman F

D Costello E K Huttley G A (2010) QIIME allows analysis of

high-throughput community sequencing data Nature Methods 7

335ndash336 httpsdoiorg101038nmethf303

Carreiro M M Sinsabaugh R L Repert D A amp Parkhurst D F

(2000) Microbial enzyme shifts explain litter decay responses to sim-

ulated nitrogen deposition Ecology 81 2359ndash2365 httpsdoiorg

1018900012-9658(2000)081[2359MESELD]20CO2

Cheeke T E Phillips R P Brzostek E R Rosling A Bever J D amp

Fransson P (2017) Dominant mycorrhizal association of trees alters

carbon and nutrient cycling by selecting for microbial groups with

distinct enzyme function New Phytologist 214 432ndash442 httpsdoi

org101111nph14343

Cheng W Parton W J Gonzalez-Meler M A Phillips R Asao S

McNickle G G Jastrow J D (2014) Synthesis and modeling

perspectives of rhizosphere priming New Phytologist 201 31ndash44

httpsdoiorg101111nph12440

Comas L H Callahan H S amp Midford P E (2014) Patterns in root

traits of woody species hosting arbuscular and ectomycorrhizas

Implications for the evolution of belowground strategies Ecology and

Evolution 4 2979ndash2990 httpsdoiorg101002ece31147

Datta R Kelkar A Baraniya D Molaei A Moulick A Meena R S amp

Formanek P (2017) Enzymatic degradation of lignin in soil A

review Sustainability 9 1163 httpsdoiorg103390su9071163

CARRARA ET AL | 11

colonization of fine roots by 559 Given the inherent limitations of

measuring root production using minirhizotron tubes or ingrowth

cores we use fine root biomass and AM colonization as proxies for

plant C allocation to gain nutrients While root turnover may be

enhanced under N fertilization we base the assumption that fine

root biomass is synonymous with belowground C allocation to fine

roots on our observations that fine root biomass was consistently

lower in the fertilized than control watersheds over all three of the

sequentially cored soil sampling dates during the growing season In

addition AM fungi have been shown to act as conduits for photo-

synthate C to rhizosphere microbes and as such represent an impor-

tant belowground flux of C (Kaiser et al 2015) Thus reduced

investment in AM colonization may also lead to reduced transfers of

photosynthate C to the rhizosphere resulting in an overall decline in

belowground C allocation

By contrast in the OH standing fine root biomass was larger in

the N-fertilized watershed although root biomass in the OH horizon

is markedly lower than in the bulk mineral soil (Table 1) However

like the bulk mineral soil AM colonization of OH roots was signifi-

cantly higher in the reference watershed (728 colonized) than the

N-fertilized watershed (609 colonized)

32 | Extracellular enzyme activity

Nearly all hydrolytic enzyme activities were lower under elevated N

conditions across all three soil fractions BG activity was significantly

lower in the N-fertilized watershed in both the bulk mineral (52

decrease) and OH (17 decrease) soil fractions (p lt 05 Figure 1a)

N fertilization significantly lowered AP activity in the bulk (12) and

rhizosphere (36) fractions of the mineral soil (p lt 05 Figure 1b)

Similarly N amendment significantly lowered NAG activity in both

bulk and rhizosphere fractions of the mineral soil by 41 and 37

respectively (p lt 05 Figure 1c) However BG activity in the rhizo-

sphere of the mineral soil was not significantly different between

0

04

08

12

16

Bulk Rhizo

AP

(m

olmiddot[

gdr

yso

il]ndash1

middot hr

ndash1)

0

1

2

3

4

5

OH0

01

02

03

04

Bulk Rhizo

BG

(m

olmiddot[g

dry

soi

l]ndash1

middot hr

ndash1)

0

05

1

15

2

OH

0

003

006

009

012

Bulk Rhizo

NA

G (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)

0

02

04

06

08

1

OH

0

04

08

12

16

Bulk Rhizo0

10

20

30

40

50

OH

0

025

05

075

1

Bulk Rhizo0

1

2

3

OH

0

1

2

3

4

5

Bulk Rhizo

Pero

xida

se (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)

0

1

2

3

4

OH

(a) (b)

(c) (d)

(e) (f)

Phen

ol o

xida

se (

mol

middot[g

dry

soil]

ndash1 middot

hrndash1

)Pr

oteo

lysi

s (

gA

A-N

middot[g

dry

soil]

ndash1 middot

hrndash1

)

Reference

+N

F IGURE 1 N fertilization reduceshydrolytic and oxidative enzyme activitiesValues are the overall seasonal meanenzyme activities (mean SE) of (a) BG(b) AP (c) NAG (d) phenol oxidase (e)peroxidase and (f) proteolysis for each soilfraction (ie bulk rhizosphere and organichorizon) measured in June July andAugust 2015 across all plots (n = 10 plotsper watershed) Asterisks indicatesignificant differences in enzyme activitybetween watersheds within soil fractions(p lt 05) Note difference in scale betweenOH vs rhizosphere and bulk soil fractionsAA-N is amino acid nitrogen

6 | CARRARA ET AL

watersheds In addition AP and NAG activities in the OH horizon

did not vary by treatment

Liginolytic oxidative enzyme activities were also generally lower

in the N-fertilized watershed Phenol oxidase activity was signifi-

cantly lower in bulk (45 decrease) rhizosphere (49 decrease) and

OH horizons (57 decrease) of the fertilized watershed relative to

the reference watershed (p lt 05 Figure 1d) Peroxidase activity was

lower in the fertilized watershed in the bulk rhizosphere and OH

soil fractions with reductions of 30 25 and 36 respectively

(p lt 05 Figure 1e) Proteolytic enzyme activity was consistently

lower in each soil fraction under N fertilization with a 48 decrease

in bulk 56 decrease in rhizosphere and 40 decrease in OH

proteolysis (Figure 1f)

33 | Microbial community composition

When fungal taxonomic units were aggregated to the phylum level

there were no significant changes in the relative abundance of the

four most common fungal phyla or the unclassified group in any soil

horizon (Figure S1) When bacterial taxonomic units were aggregated

to the phylum level there were limited shifts in the relative abun-

dance of the seven most common bacterial phyla Relative abun-

dance of Actinobacteria was higher in the OH soil of the fertilized

watershed (Figure S2a) Relative abundance of Proteobacteria was

lower in the fertilized bulk soil and relative abundance of Firmicutes

was higher in both rhizosphere and bulk soil in the fertilized water-

shed (Figure S2bc)

Adonis analysis of bacterial communities revealed significant

effects for watershed soil horizon (p lt 001) and their interaction

(p = 02 total model R2 = 32) Within watersheds post hoc compar-

isons showed OH communities were different than bulk and rhizo-

sphere communities (Figure S3ab) There was no difference

between bulk and rhizosphere communities (Figure S3ab) Across

watersheds bacterial community composition differed in all soil frac-

tions such that OH rhizosphere and bulk soil exhibited unique com-

munities in the N-fertilized watershed compared to the reference

(Figure 2ace)

Adonis analysis of fungal communities revealed significant effects

of watershed and soil horizon (p lt 001 total model R2 = 12) but

not their interaction Within watersheds post hoc comparisons of

fungal communities showed OH communities were different than

bulk and rhizosphere communities in both watersheds but there was

no difference between bulk and rhizosphere communities

(Figure S3cd) Across watersheds fungal community composition

within OH and bulk soil fractions was significantly different (p lt 01

Figure 2bd) However there was no significant difference in fungal

communities between watersheds in rhizosphere soil (Figure 2f)

Comparison of bacterial and enzymatic NMDS scores across all

soil horizons showed a positive relationship between bacterial com-

munities and enzyme profiles (R2 = 48 p lt 01 Figure S4a) Compar-

ison of fungal and enzymatic NMDS scores across all soil horizons and

both watersheds showed no significant relationship between fungal

communities and enzyme profiles (Figure S4b) Across watersheds

comparison of bacterial and enzymatic NMDS scores showed a nega-

tive relationship between bacterial communities and enzyme profiles

in the bulk soil (R2 = 36 p lt 05 Figure 3a) but no significant rela-

tionship in rhizosphere soil Comparison of fungal and enzymatic

NMDS scores across watersheds showed no significant relationships

in either bulk or rhizosphere soil (Figure 3b) Linear regression of

AM colonization and the first bacterial NMDS axis resulted in a posi-

tive linear relationship in bulk soil (R2 = 063 p lt 001 Figure 3c) but

not rhizosphere soil There were no significant linear relationships

between AM colonization and the first fungal NMDS axis in either

soil fraction (Figure 3d)

34 | Net N mineralization and nitrification

The only significant difference we found in rates of nitrogen cycling

was that N mineralization and net nitrification in the OH were 40

and 51 higher in the fertilized watershed than the reference N

transformation rates did not significantly differ between watersheds

in either the bulk or rhizosphere soil fractions (Figure 4ab)

35 | Modeling potential impacts of plantndashmicrobiallinkages on soil decomposition

When we ran the model to steady state we found that reductions

in root biomass and root C transfers to the rhizosphere in the N-fer-

tilized watershed reduced microbial enzyme pools by ~16 and

enhanced soil C by ~3 compared to the reference watershed

(Table 2) Even though exudation rates on a per g root basis were

the same the fertilized watershed had lower overall exudations rates

at the ecosystem scale than the control watershed because of its

lower root biomass (Table 2) When we reduced exudation rates by

25 on a per g root basis in the fertilized watershed there was a

further exacerbation in the reduction in microbial enzyme activity to

a ~28 decline and a larger increase in soil C by ~20 (Table 2)

4 | DISCUSSION

Here we provide evidence that coupled interactions among plants

fungi and bacteria play an important role in enzyme activity

responses to N fertilization For fungi we observed distinct shifts in

fungal community structure in response to N fertilization (Figure 2b

d) but we found no evidence for a link between these shifts and

extracellular enzyme activity (Figure 3b) By contrast we found that

bacterial community composition shifts under N fertilization are cor-

related with declines in enzyme activity (Figure 3a) and that these

compositional shifts are tightly coupled to reductions in plant C allo-

cation to roots and mycorrhizal fungi (Figure 3c) Overall these

results suggest that N fertilization drives an integrated ecosystem

response whereby reductions in plant C allocation to roots and AM

fungi feedback on bacterial community structure and function

While whole-watershed fertilization at the Fernow results in a

pseudoreplicated experimental design (Hurlbert 1984) we conclude

CARRARA ET AL | 7

that the effects we measured are driven by N fertilization rather

than pre-existing differences between these adjacent watersheds

for four main reasons First soil chemistry (ie soil pH cation

exchange capacity nutrient content etc) were similar at the begin-

ning of the experiment (Adams amp Angradi 1996) Second the

amount of N added to the watershed yearly was originally chosen in

1989 to approximately double ambient N deposition rates but is

now more than quadruple current rates and as such it seems unli-

kely that this does not incur a biogeochemical response Third the

results from this watershed study are consistent with measurements

we made during the same year in a replicated N fertilization study

lt2 km away from these watersheds (the Fork Mountain Long-Term

Soil Productivity Plots Adams Burger Zelazny amp Baumgras 2004)

In this small-scale replicated study N fertilization reduced fine root

biomass and ligninolytic enzyme activity (Figure S5) Finally this

work builds upon other research at the Fernow that has found the

N-fertilized watershed had lower rates of litter decomposition

(Adams amp Angradi 1996) reduced understory richness (Gilliam et al

1994 2016 Walter Adams Gilliam amp Peterjohn 2017) and altered

N cycling (Adams et al 1993 Burnham Cumming Adams amp Peter-

john 2017 Gilliam Yurish amp Adams 2001 Gilliam et al 1996

2016) compared to the reference watershed

While we did observe significant declines in enzyme activity

across all three soil fractions particularly for the liginolytic enzymes

(Figure 1andashf) these declines were not correlated with significant

shifts in fungal community composition (Figure 3b) The lack of a

clear link between enzyme declines and changes in the fungal com-

munity does not support the prevailing paradigm that white-rot

Basidiomycota are the dominant cause of a decline in enzyme activi-

ties following N additions (Edwards Zak Kellner Eisenlord amp Pregit-

zer 2011 Fog 1988 Freedman Romanowicz Upchurch amp Zak

2015 Morrison et al 2016) It is possible that fungal enzyme activ-

ity response to N fertilization is independent of community composi-

tion (ie investment in enzyme activity declines with no change in

community structure) however microbial community composition

has been linked to catabolic functioning and enzyme activities across

N gradients and seasons (Fierer et al 2011 Vorıskova Brabcova

Cajthaml amp Baldrian 2014) Furthermore our data indicate that

ndash008

0

008

016

024

032

ndash09 ndash06 ndash03 0 03 06

Rhi

zo f

ungi

NM

DS2

Rhizo fungi NMDS1

ndash04

ndash02

0

02

04

ndash02 ndash01 0 01 02 03 04

Rhi

zo b

acte

ria

NM

DS2

Rhizo bacteria NMDS1

ndash02

ndash01

0

01

02

03

ndash02 0 02 04 06

Bul

k ba

cter

ia N

MD

S2

Bulk bacteria NMDS1

ndash04

ndash032

ndash024

ndash016

ndash008

0

ndash04 ndash02 0 02 04

OH

fun

gi N

MD

S2

OH fungi NMDS1

ndash06

ndash04

ndash02

0

02

ndash05 ndash04 ndash03 ndash02 ndash01 0

OH

bac

teri

a N

MD

S2

OH bacteria NMDS1

Reference

+N

ndash02

ndash01

0

01

02

03

04

ndash06 ndash04 ndash02 0 02 04

Bul

k fu

ngi N

MD

S2

Bulk fungi NMDS1

(a) (b)

(c) (d)

(e) (f)

plt01

plt01

plt01

plt01

plt01

F IGURE 2 N fertilization alteredbacterial community composition in OH (a)bulk (c) and rhizosphere (e) soils andfungal community composition in OH (b)and bulk (d) soils but not rhizosphere (f)All community data were obtained for eachsoil fraction in July 2015 (n = 10 plots perwatershed) p values indicate significantdifferences between the N-fertilized andreference community

8 | CARRARA ET AL

shifts in bacterial community composition under elevated N are cor-

related with reduced enzyme activity at the Fernow (Figure 3a)

While changes in bacterial community may be influenced by overall

fungal community composition these shifts in bacterial community

composition are tightly coupled to AM colonization (a metric of

belowground C allocation) suggesting that plant responses to N fer-

tilization feedback on bacterial community structure and function

(Figure 3c) While we cannot rule out that N-induced declines in

total fungal biomass led to reductions in liginolytic enzyme activity

(DeForest et al 2004 Frey et al 2004 Treseder 2008 Wallenstein

et al 2006) the dominance of AM trees in our plots whose inor-

ganic nutrient economy is largely driven by bacteria suggests that

free-living fungi may not be an important driver of N deposition

responses in AM-dominated systems (Cheeke et al 2017 Phillips

Brzostek amp Midgley 2013)

In contrast it appears that the declines in enzyme activity we

observed appear to be the result of a cascade of ecosystem

responses affecting both microbial community composition and plant

C allocation belowground The N-induced declines in fine root bio-

mass AM colonization and root morphology all indicate that there

was a reduction in the investment of C belowground by trees to gain

nutrients (Table 1) While previous research has shown that below-

ground C allocation is inversely correlated with N availability (Bae

Fahey Yanai amp Fisk 2015 Haynes amp Gower 1995 Phillips amp Fahey

2005) the tight linkage between the first bacterial NMDS axis and

AM colonization suggest that N-induced declines in belowground

C allocation caused a shift in the bacterial community composition

and subsequent reductions in enzyme activity (Figure 3ac) This

result also supports the emerging view that roots are active engi-

neers of microbial activity (Brzostek et al 2013 Finzi et al 2015

Yin et al 2014) and highlights the need to integrate root-microbial

interactions into our conceptual and predictive modeling frameworks

of how forest ecosystems respond to global change (Johnson

Gehring amp Jansa 2016 Terrer Vicca Hungate Phillips amp Prentice

2016)

An additional impact of the N fertilization treatment is that it

reduced soil pH (as measured in a 001 M calcium chloride buffer) of

the upper 5 cm of mineral soil in the fertilized watershed to 34

compared to 38 in the control watershed (Peterjohn unpublished

data) Soil pH is an important control on microbial community diver-

sity (Fierer et al 2012 Kaiser et al 2016 Lauber Hamady Knight

amp Fierer 2009) microbial biomass and enzyme activities (Rousk amp

Baath 2011 Sinsabaugh 2010) Thus the decline in soil pH may

account for a portion of the enzyme reductions we observed under

N fertilization To address this we assayed the sensitivity of phenol

oxidase and peroxidase enzyme activity in organic horizon bulk and

rhizosphere soils from the control watershed to three different levels

of pH that spanned a 1 unit shift (data not shown) Peroxidase activ-

ity was insensitive to pH (slope = 074 r2 = 05 p gt 05) While

phenol oxidase activity did significantly increase as pH increased

(slope = 010 r2 = 053 p lt 05) this sensitivity would only account

for a 10 decline in activity Given that we observed a 50 decline

in phenol oxidase in the treatment watershed coupled with the

greater overall importance of peroxidase enzymes in our study (ie

nearly an order of magnitude higher activity) it appears that pH is

an important but secondary driver of the enzyme activity responses

we observed

Over longer time scales the reductions in root and microbial

activity we observed at the Fernow may have important implications

for soil C stocks In our model simulation we found that feedbacks

between reductions in root biomass and enzyme production have

the potential to drive nearly a 3 increase in soil C stocks (Table 2)

When these were coupled with a 25 reduction in specific root C

exudation rates soil C in the fertilized watershed increased by nearly

20 over the 30-yr simulation This model was designed to be theo-

retical and as such it is used here to show the sensitivity of the

y = 0004x ndash 007R = 63

ndash02

0

02

04

06

0 25 50 75 100Bul

k B

acte

ria

OT

U N

MD

S1

AM Colonization ()

y = ndash0159x + 013R = 360

004

008

012

016

02

ndash02 0 02 04 06

Enz

yme

NM

DS

1

Bacteria OTU NMDS1

+NReference

0

004

008

012

016

02

ndash06 ndash04 ndash02 0 02 04

Enz

yme

NM

DS

1

Fungal OTU NMDS1

ndash06

ndash04

ndash02

0

02

04

0 25 50 75 100

Bul

k Fu

ngi

OT

U N

MD

S1

AM Colonization ()

plt05

plt001

(a)

(c)

(b)

(d)F IGURE 3 Bacterial but not fungalcommunity composition is correlated withthe first NMDS axis of enzyme activity (aamp b) Percent AM colonization is correlatedwith bacterial community composition (c)but not fungal community composition (d)Data presented are bulk soil communitycomposition data from July 2015 (n = 10plots per watershed) p values indicatesignificance of correlation

CARRARA ET AL | 9

system to the perturbations in plant inputs and is not meant to be

quantitatively predictive However this simple modeling effort pro-

vides compelling evidence that N-induced shifts in rootndashmicrobial

interactions have the potential to alter not only microbial production

of soil enzymes but also the size of the soil C pool To generate the

necessary data for fully integrating root-microbial interactions into

more sophisticated ecosystem models future research should couple

direct measurements of belowground C allocation responses to N

fertilization (eg total belowground C allocation root exudation)

with the resulting impacts on microbial activity

Given the prevailing paradigm that free-living fungi drive soil

enzyme activity and subsequent decomposition responses to N fer-

tilization (Carreiro et al 2000 Fog 1988 Waldrop et al 2004 Zak

et al 2008) our results raise an important question of why shifts in

plant C allocation bacterial community structure and enzyme activi-

ties were tightly coupled at the Fernow The divergent results

between the Fernow and other long-term fertilization sites may be

the result of differences in ambient N deposition and tree species

composition The Fernow is in a region that has historically received

some of the highest levels of N deposition in the United States

(Driscoll et al 2001) which may have enhanced bacterial dominance

before treatment began in 1989 By contrast N fertilization experi-

ments that have linked reductions in soil C decomposition to fungal

community composition and activity tend to be located in areas with

lower historical N deposition rates (Edwards et al 2011 Freedman

et al 2015 Frey et al 2004 Morrison et al 2016 Wallenstein

et al 2006) Second the Fernow is predominantly dominated by

AM trees whereas most N fertilization experiments have tended to

occur in ecosystems dominated by ECM trees with ECM-dominated

sites comprising ~80 of the studies included in the Janssens et al

(2010) meta-analysis AM trees show greater growth enhancements

with N addition (Thomas et al 2010) are characterized by lower

fungal to bacterial ratios (Cheeke et al 2017) and are less depen-

dent on plantndashmicrobial interactions to access N than ECM trees

(Brzostek Dragoni Brown amp Phillips 2015) As such in AM-domi-

nated systems like the Fernow bacteria may outweigh fungi in con-

trolling soil decomposition responses to N fertilization Additionally

evidence that ECM trees rely more on roots and rhizosphere

microbes to access N than AM trees suggests that N fertilization

may lead to greater declines in belowground C allocation and

enzyme activity than observed at the Fernow (Brzostek et al 2015

Yin et al 2014) Moving forward the equal distribution of ECM and

AM trees across the temperate forest landscape highlights a critical

need to investigate ECM and AM stand responses to N fertilization

within the same ecosystem (Midgley amp Phillips 2014)

Although enzyme declines were observed across all three soil

fractions (Figure 1andashf) N cycling shifts were only observed in the

OH where rates were elevated by nearly 50 and 40 for mineral-

ization and nitrification respectively (Figure 4ab) OH and mineral

soils differ in key biogeochemical traits and this may contribute to

these divergent N cycling responses In temperate forests OH

0

05

1

15

Bulk Rhizo

Net

N m

iner

aliz

atio

n (

gN

middotgndash1

middotday

ndash1)

Control

+N

0

3

6

9

12

15

18

OH

0

05

1

15

Bulk Rhizo

Net

nitr

ific

atio

n ((

gN

middotgndash1

middotday

ndash1)

0

2

4

6

8

10

12

OH

(a)

(b)

Reference

F IGURE 4 N fertilization increases (a) N mineralization and (b)nitrification in the OH but not the mineral soil Values are theoverall watershed-level mean rates (mean SE) measured in JuneJuly and August 2015 for each soil fraction (n = 10 plots perwatershed) Asterisks indicate significant differences betweentreatments (p lt 05)

TABLE 2 At model steady state fertilization reduced microbialenzyme pools by ~16 and enhanced soil C by ~3

Watershed ExudationEnzyme pool(mgcm3)

Soil C pool(mgcm3)

Control No change 00136 18473

Fertilized No change 00114 19092

25 reduction 00097 22116

When root C exudation was reduced 25 on a per g root basis in the

fertilized watershed there was a further exacerbation in the reduction in

microbial enzyme activity to a ~28 decline and a larger increase in soil

C by ~20

10 | CARRARA ET AL

typically have rapid N cycling rates reflecting greater organic matter

content and root densities than mineral soils (Brzostek amp Finzi

2011) Shifts in the ratio of gross immobilization to gross mineraliza-

tion may also play a role Net N mineralization rates increased in the

OH despite N fertilization induced declines in proteolytic and chiti-

nolytic enzyme activity that produce N monomers for microbial

uptake While we do not have data on gross N cycling we hypothe-

size that declines in gross microbial N immobilization due to reduced

N demand may have outpaced declines in gross N mineralization in

the fertilized watershed Regardless of the exact mechanism it is

important to put the OH results into context on a mass per unit

area basis the OH is less important to total ecosystem N cycling

than the underlying mineral soil

Much of the research on soil N fertilization has focused on

aboveground responses such as litter input and quality or fungal

community shifts to explain widely observed reductions in enzyme

activities and subsequent soil decomposition (Edwards et al 2011

Frey et al 2004 Morrison et al 2016 Sinsabaugh et al 2008

Waldrop et al 2004 Wallenstein et al 2006) Our results provide

evidence that enzyme activity declines under long-term N fertiliza-

tion at the Fernow are driven by a cascade of ecosystem responses

whereby reductions in belowground plant C investment lead to

shifts in bacterial community structure and a decline in their ability

to degrade soil organic matter Thus there is a need to integrate

plantndashmicrobial interactions into our current conceptual and predic-

tive models of N deposition impacts on temperate forests in order

to forecast soil C responses to shifting N deposition regimes

ACKNOWLEDGEMENTS

We acknowledge Mary Beth Adams Tom Schuler and the US Forest

Service staff at the Fernow Experimental Forest for logistical assis-

tance and access to the experimental watersheds and the LTSP site

This work was also supported by the National Science Foundation

Graduate Research Fellowship to Joseph Carrara under Grant No

DGE-1102689 and by the Long-Term Research in Environmental

Biology (LTREB) program at the National Science Foundation (Grant

Nos DEB-0417678 and DEB-1019522) to William Peterjohn Colin

Averill was supported by the NOAA Climate and Global Change

Postdoctoral Fellowship Program administered by Cooperative Pro-

grams for the Advancement of Earth System Science (CPAESS)

University Corporation for Atmospheric Research (UCAR) Boulder

Colorado USA We acknowledge the WVU Genomics Core Facility

Morgantown WV for support provided to help make this publication

possible We also thank Leah Baldinger Brittany Carver Mark Burn-

ham Hannah Hedrick Jennifer Mangano and Catherine Sesa for

assistance in the field and in the laboratory

ORCID

Joseph E Carrara httporcidorg0000-0003-0597-1175

Colin Averill httporcidorg0000-0003-4035-7760

REFERENCES

Adams M B amp Angradi T R (1996) Decomposition and nutrient

dynamics of hardwood leaf litter in the Femow Whole-Watershed

Acidification Experiment Forest Ecology and Management 1127 61ndash

69 httpsdoiorg1010160378-1127(95)03695-4

Adams M B Burger J Zelazny L amp Baumgras J (2004) Descrip-

tion of the fork mountain long-term soil productivity study Site

characterization USDA Forest Service General Technical Report NE

43 323

Adams M B Edwards P J Wood F amp Kochenderfer J N (1993)

Artificial watershed acidification on the Fernow Experimental Forest

USA Journal of Hydrology 150 505ndash519 httpsdoiorg101016

0022-1694(93)90123-Q

Allison S D Wallenstein M D amp Bradford M A (2010) Soil-carbon

response to warming dependent on microbial physiology Nature Geo-

science 3 336ndash340 httpsdoiorg101038ngeo846

Bae K Fahey T J Yanai R D amp Fisk M (2015) Soil nitrogen avail-

ability affects belowground carbon allocation and soil respiration in

northern hardwood forests of new hampshire Ecosystems 18 1179ndash

1191 httpsdoiorg101007s10021-015-9892-7

Brzostek E R Dragoni D Brown Z A amp Phillips R P (2015) Mycor-

rhizal type determines the magnitude and direction of root-induced

changes in decomposition in a temperate forest New Phytologist

206 1274ndash1282 httpsdoiorg101111nph13303

Brzostek E R amp Finzi A C (2011) Substrate supply fine roots and

temperature control proteolytic enzyme activity in temperate forest

soils Ecology 92 892ndash902 httpsdoiorg10189010-18031

Brzostek E R Fisher J B amp Phillips R P (2014) Modeling the carbon

cost of plant nitrogen acquisition Mycorrhizal trade-offs and multi-

path resistance uptake improve predictions of retranslocation Journal

of Geophysical Research Biogeosciences 119 1684ndash1697

Brzostek E R Greco A Drake J E amp Finzi A C (2013) Root carbon

inputs to the rhizosphere stimulate extracellular enzyme activity and

increase nitrogen availability in temperate forest soils Biogeochem-

istry 115 65ndash76 httpsdoiorg101007s10533-012-9818-9

Burnham M B Cumming J R Adams M B amp Peterjohn W T

(2017) Soluble soil aluminum alters the relative uptake of mineral

nitrogen forms by six mature temperate broadleaf tree species

possible implications for watershed nitrate retention Oecologia

185 1ndash11

Caporaso J G Kuczynski J Stombaugh J Bittinger K Bushman F

D Costello E K Huttley G A (2010) QIIME allows analysis of

high-throughput community sequencing data Nature Methods 7

335ndash336 httpsdoiorg101038nmethf303

Carreiro M M Sinsabaugh R L Repert D A amp Parkhurst D F

(2000) Microbial enzyme shifts explain litter decay responses to sim-

ulated nitrogen deposition Ecology 81 2359ndash2365 httpsdoiorg

1018900012-9658(2000)081[2359MESELD]20CO2

Cheeke T E Phillips R P Brzostek E R Rosling A Bever J D amp

Fransson P (2017) Dominant mycorrhizal association of trees alters

carbon and nutrient cycling by selecting for microbial groups with

distinct enzyme function New Phytologist 214 432ndash442 httpsdoi

org101111nph14343

Cheng W Parton W J Gonzalez-Meler M A Phillips R Asao S

McNickle G G Jastrow J D (2014) Synthesis and modeling

perspectives of rhizosphere priming New Phytologist 201 31ndash44

httpsdoiorg101111nph12440

Comas L H Callahan H S amp Midford P E (2014) Patterns in root

traits of woody species hosting arbuscular and ectomycorrhizas

Implications for the evolution of belowground strategies Ecology and

Evolution 4 2979ndash2990 httpsdoiorg101002ece31147

Datta R Kelkar A Baraniya D Molaei A Moulick A Meena R S amp

Formanek P (2017) Enzymatic degradation of lignin in soil A

review Sustainability 9 1163 httpsdoiorg103390su9071163

CARRARA ET AL | 11

watersheds In addition AP and NAG activities in the OH horizon

did not vary by treatment

Liginolytic oxidative enzyme activities were also generally lower

in the N-fertilized watershed Phenol oxidase activity was signifi-

cantly lower in bulk (45 decrease) rhizosphere (49 decrease) and

OH horizons (57 decrease) of the fertilized watershed relative to

the reference watershed (p lt 05 Figure 1d) Peroxidase activity was

lower in the fertilized watershed in the bulk rhizosphere and OH

soil fractions with reductions of 30 25 and 36 respectively

(p lt 05 Figure 1e) Proteolytic enzyme activity was consistently

lower in each soil fraction under N fertilization with a 48 decrease

in bulk 56 decrease in rhizosphere and 40 decrease in OH

proteolysis (Figure 1f)

33 | Microbial community composition

When fungal taxonomic units were aggregated to the phylum level

there were no significant changes in the relative abundance of the

four most common fungal phyla or the unclassified group in any soil

horizon (Figure S1) When bacterial taxonomic units were aggregated

to the phylum level there were limited shifts in the relative abun-

dance of the seven most common bacterial phyla Relative abun-

dance of Actinobacteria was higher in the OH soil of the fertilized

watershed (Figure S2a) Relative abundance of Proteobacteria was

lower in the fertilized bulk soil and relative abundance of Firmicutes

was higher in both rhizosphere and bulk soil in the fertilized water-

shed (Figure S2bc)

Adonis analysis of bacterial communities revealed significant

effects for watershed soil horizon (p lt 001) and their interaction

(p = 02 total model R2 = 32) Within watersheds post hoc compar-

isons showed OH communities were different than bulk and rhizo-

sphere communities (Figure S3ab) There was no difference

between bulk and rhizosphere communities (Figure S3ab) Across

watersheds bacterial community composition differed in all soil frac-

tions such that OH rhizosphere and bulk soil exhibited unique com-

munities in the N-fertilized watershed compared to the reference

(Figure 2ace)

Adonis analysis of fungal communities revealed significant effects

of watershed and soil horizon (p lt 001 total model R2 = 12) but

not their interaction Within watersheds post hoc comparisons of

fungal communities showed OH communities were different than

bulk and rhizosphere communities in both watersheds but there was

no difference between bulk and rhizosphere communities

(Figure S3cd) Across watersheds fungal community composition

within OH and bulk soil fractions was significantly different (p lt 01

Figure 2bd) However there was no significant difference in fungal

communities between watersheds in rhizosphere soil (Figure 2f)

Comparison of bacterial and enzymatic NMDS scores across all

soil horizons showed a positive relationship between bacterial com-

munities and enzyme profiles (R2 = 48 p lt 01 Figure S4a) Compar-

ison of fungal and enzymatic NMDS scores across all soil horizons and

both watersheds showed no significant relationship between fungal

communities and enzyme profiles (Figure S4b) Across watersheds

comparison of bacterial and enzymatic NMDS scores showed a nega-

tive relationship between bacterial communities and enzyme profiles

in the bulk soil (R2 = 36 p lt 05 Figure 3a) but no significant rela-

tionship in rhizosphere soil Comparison of fungal and enzymatic

NMDS scores across watersheds showed no significant relationships

in either bulk or rhizosphere soil (Figure 3b) Linear regression of

AM colonization and the first bacterial NMDS axis resulted in a posi-

tive linear relationship in bulk soil (R2 = 063 p lt 001 Figure 3c) but

not rhizosphere soil There were no significant linear relationships

between AM colonization and the first fungal NMDS axis in either

soil fraction (Figure 3d)

34 | Net N mineralization and nitrification

The only significant difference we found in rates of nitrogen cycling

was that N mineralization and net nitrification in the OH were 40

and 51 higher in the fertilized watershed than the reference N

transformation rates did not significantly differ between watersheds

in either the bulk or rhizosphere soil fractions (Figure 4ab)

35 | Modeling potential impacts of plantndashmicrobiallinkages on soil decomposition

When we ran the model to steady state we found that reductions

in root biomass and root C transfers to the rhizosphere in the N-fer-

tilized watershed reduced microbial enzyme pools by ~16 and

enhanced soil C by ~3 compared to the reference watershed

(Table 2) Even though exudation rates on a per g root basis were

the same the fertilized watershed had lower overall exudations rates

at the ecosystem scale than the control watershed because of its

lower root biomass (Table 2) When we reduced exudation rates by

25 on a per g root basis in the fertilized watershed there was a

further exacerbation in the reduction in microbial enzyme activity to

a ~28 decline and a larger increase in soil C by ~20 (Table 2)

4 | DISCUSSION

Here we provide evidence that coupled interactions among plants

fungi and bacteria play an important role in enzyme activity

responses to N fertilization For fungi we observed distinct shifts in

fungal community structure in response to N fertilization (Figure 2b

d) but we found no evidence for a link between these shifts and

extracellular enzyme activity (Figure 3b) By contrast we found that

bacterial community composition shifts under N fertilization are cor-

related with declines in enzyme activity (Figure 3a) and that these

compositional shifts are tightly coupled to reductions in plant C allo-

cation to roots and mycorrhizal fungi (Figure 3c) Overall these

results suggest that N fertilization drives an integrated ecosystem

response whereby reductions in plant C allocation to roots and AM

fungi feedback on bacterial community structure and function

While whole-watershed fertilization at the Fernow results in a

pseudoreplicated experimental design (Hurlbert 1984) we conclude

CARRARA ET AL | 7

that the effects we measured are driven by N fertilization rather

than pre-existing differences between these adjacent watersheds

for four main reasons First soil chemistry (ie soil pH cation

exchange capacity nutrient content etc) were similar at the begin-

ning of the experiment (Adams amp Angradi 1996) Second the

amount of N added to the watershed yearly was originally chosen in

1989 to approximately double ambient N deposition rates but is

now more than quadruple current rates and as such it seems unli-

kely that this does not incur a biogeochemical response Third the

results from this watershed study are consistent with measurements

we made during the same year in a replicated N fertilization study

lt2 km away from these watersheds (the Fork Mountain Long-Term

Soil Productivity Plots Adams Burger Zelazny amp Baumgras 2004)

In this small-scale replicated study N fertilization reduced fine root

biomass and ligninolytic enzyme activity (Figure S5) Finally this

work builds upon other research at the Fernow that has found the

N-fertilized watershed had lower rates of litter decomposition

(Adams amp Angradi 1996) reduced understory richness (Gilliam et al

1994 2016 Walter Adams Gilliam amp Peterjohn 2017) and altered

N cycling (Adams et al 1993 Burnham Cumming Adams amp Peter-

john 2017 Gilliam Yurish amp Adams 2001 Gilliam et al 1996

2016) compared to the reference watershed

While we did observe significant declines in enzyme activity

across all three soil fractions particularly for the liginolytic enzymes

(Figure 1andashf) these declines were not correlated with significant

shifts in fungal community composition (Figure 3b) The lack of a

clear link between enzyme declines and changes in the fungal com-

munity does not support the prevailing paradigm that white-rot

Basidiomycota are the dominant cause of a decline in enzyme activi-

ties following N additions (Edwards Zak Kellner Eisenlord amp Pregit-

zer 2011 Fog 1988 Freedman Romanowicz Upchurch amp Zak

2015 Morrison et al 2016) It is possible that fungal enzyme activ-

ity response to N fertilization is independent of community composi-

tion (ie investment in enzyme activity declines with no change in

community structure) however microbial community composition

has been linked to catabolic functioning and enzyme activities across

N gradients and seasons (Fierer et al 2011 Vorıskova Brabcova

Cajthaml amp Baldrian 2014) Furthermore our data indicate that

ndash008

0

008

016

024

032

ndash09 ndash06 ndash03 0 03 06

Rhi

zo f

ungi

NM

DS2

Rhizo fungi NMDS1

ndash04

ndash02

0

02

04

ndash02 ndash01 0 01 02 03 04

Rhi

zo b

acte

ria

NM

DS2

Rhizo bacteria NMDS1

ndash02

ndash01

0

01

02

03

ndash02 0 02 04 06

Bul

k ba

cter

ia N

MD

S2

Bulk bacteria NMDS1

ndash04

ndash032

ndash024

ndash016

ndash008

0

ndash04 ndash02 0 02 04

OH

fun

gi N

MD

S2

OH fungi NMDS1

ndash06

ndash04

ndash02

0

02

ndash05 ndash04 ndash03 ndash02 ndash01 0

OH

bac

teri

a N

MD

S2

OH bacteria NMDS1

Reference

+N

ndash02

ndash01

0

01

02

03

04

ndash06 ndash04 ndash02 0 02 04

Bul

k fu

ngi N

MD

S2

Bulk fungi NMDS1

(a) (b)

(c) (d)

(e) (f)

plt01

plt01

plt01

plt01

plt01

F IGURE 2 N fertilization alteredbacterial community composition in OH (a)bulk (c) and rhizosphere (e) soils andfungal community composition in OH (b)and bulk (d) soils but not rhizosphere (f)All community data were obtained for eachsoil fraction in July 2015 (n = 10 plots perwatershed) p values indicate significantdifferences between the N-fertilized andreference community

8 | CARRARA ET AL

shifts in bacterial community composition under elevated N are cor-

related with reduced enzyme activity at the Fernow (Figure 3a)

While changes in bacterial community may be influenced by overall

fungal community composition these shifts in bacterial community

composition are tightly coupled to AM colonization (a metric of

belowground C allocation) suggesting that plant responses to N fer-

tilization feedback on bacterial community structure and function

(Figure 3c) While we cannot rule out that N-induced declines in

total fungal biomass led to reductions in liginolytic enzyme activity

(DeForest et al 2004 Frey et al 2004 Treseder 2008 Wallenstein

et al 2006) the dominance of AM trees in our plots whose inor-

ganic nutrient economy is largely driven by bacteria suggests that

free-living fungi may not be an important driver of N deposition

responses in AM-dominated systems (Cheeke et al 2017 Phillips

Brzostek amp Midgley 2013)

In contrast it appears that the declines in enzyme activity we

observed appear to be the result of a cascade of ecosystem

responses affecting both microbial community composition and plant

C allocation belowground The N-induced declines in fine root bio-

mass AM colonization and root morphology all indicate that there

was a reduction in the investment of C belowground by trees to gain

nutrients (Table 1) While previous research has shown that below-

ground C allocation is inversely correlated with N availability (Bae

Fahey Yanai amp Fisk 2015 Haynes amp Gower 1995 Phillips amp Fahey

2005) the tight linkage between the first bacterial NMDS axis and

AM colonization suggest that N-induced declines in belowground

C allocation caused a shift in the bacterial community composition

and subsequent reductions in enzyme activity (Figure 3ac) This

result also supports the emerging view that roots are active engi-

neers of microbial activity (Brzostek et al 2013 Finzi et al 2015

Yin et al 2014) and highlights the need to integrate root-microbial

interactions into our conceptual and predictive modeling frameworks

of how forest ecosystems respond to global change (Johnson

Gehring amp Jansa 2016 Terrer Vicca Hungate Phillips amp Prentice

2016)

An additional impact of the N fertilization treatment is that it

reduced soil pH (as measured in a 001 M calcium chloride buffer) of

the upper 5 cm of mineral soil in the fertilized watershed to 34

compared to 38 in the control watershed (Peterjohn unpublished

data) Soil pH is an important control on microbial community diver-

sity (Fierer et al 2012 Kaiser et al 2016 Lauber Hamady Knight

amp Fierer 2009) microbial biomass and enzyme activities (Rousk amp

Baath 2011 Sinsabaugh 2010) Thus the decline in soil pH may

account for a portion of the enzyme reductions we observed under

N fertilization To address this we assayed the sensitivity of phenol

oxidase and peroxidase enzyme activity in organic horizon bulk and

rhizosphere soils from the control watershed to three different levels

of pH that spanned a 1 unit shift (data not shown) Peroxidase activ-

ity was insensitive to pH (slope = 074 r2 = 05 p gt 05) While

phenol oxidase activity did significantly increase as pH increased

(slope = 010 r2 = 053 p lt 05) this sensitivity would only account

for a 10 decline in activity Given that we observed a 50 decline

in phenol oxidase in the treatment watershed coupled with the

greater overall importance of peroxidase enzymes in our study (ie

nearly an order of magnitude higher activity) it appears that pH is

an important but secondary driver of the enzyme activity responses

we observed

Over longer time scales the reductions in root and microbial

activity we observed at the Fernow may have important implications

for soil C stocks In our model simulation we found that feedbacks

between reductions in root biomass and enzyme production have

the potential to drive nearly a 3 increase in soil C stocks (Table 2)

When these were coupled with a 25 reduction in specific root C

exudation rates soil C in the fertilized watershed increased by nearly

20 over the 30-yr simulation This model was designed to be theo-

retical and as such it is used here to show the sensitivity of the

y = 0004x ndash 007R = 63

ndash02

0

02

04

06

0 25 50 75 100Bul

k B

acte

ria

OT

U N

MD

S1

AM Colonization ()

y = ndash0159x + 013R = 360

004

008

012

016

02

ndash02 0 02 04 06

Enz

yme

NM

DS

1

Bacteria OTU NMDS1

+NReference

0

004

008

012

016

02

ndash06 ndash04 ndash02 0 02 04

Enz

yme

NM

DS

1

Fungal OTU NMDS1

ndash06

ndash04

ndash02

0

02

04

0 25 50 75 100

Bul

k Fu

ngi

OT

U N

MD

S1

AM Colonization ()

plt05

plt001

(a)

(c)

(b)

(d)F IGURE 3 Bacterial but not fungalcommunity composition is correlated withthe first NMDS axis of enzyme activity (aamp b) Percent AM colonization is correlatedwith bacterial community composition (c)but not fungal community composition (d)Data presented are bulk soil communitycomposition data from July 2015 (n = 10plots per watershed) p values indicatesignificance of correlation

CARRARA ET AL | 9

system to the perturbations in plant inputs and is not meant to be

quantitatively predictive However this simple modeling effort pro-

vides compelling evidence that N-induced shifts in rootndashmicrobial

interactions have the potential to alter not only microbial production

of soil enzymes but also the size of the soil C pool To generate the

necessary data for fully integrating root-microbial interactions into

more sophisticated ecosystem models future research should couple

direct measurements of belowground C allocation responses to N

fertilization (eg total belowground C allocation root exudation)

with the resulting impacts on microbial activity

Given the prevailing paradigm that free-living fungi drive soil

enzyme activity and subsequent decomposition responses to N fer-

tilization (Carreiro et al 2000 Fog 1988 Waldrop et al 2004 Zak

et al 2008) our results raise an important question of why shifts in

plant C allocation bacterial community structure and enzyme activi-

ties were tightly coupled at the Fernow The divergent results

between the Fernow and other long-term fertilization sites may be

the result of differences in ambient N deposition and tree species

composition The Fernow is in a region that has historically received

some of the highest levels of N deposition in the United States

(Driscoll et al 2001) which may have enhanced bacterial dominance

before treatment began in 1989 By contrast N fertilization experi-

ments that have linked reductions in soil C decomposition to fungal

community composition and activity tend to be located in areas with

lower historical N deposition rates (Edwards et al 2011 Freedman

et al 2015 Frey et al 2004 Morrison et al 2016 Wallenstein

et al 2006) Second the Fernow is predominantly dominated by

AM trees whereas most N fertilization experiments have tended to

occur in ecosystems dominated by ECM trees with ECM-dominated

sites comprising ~80 of the studies included in the Janssens et al

(2010) meta-analysis AM trees show greater growth enhancements

with N addition (Thomas et al 2010) are characterized by lower

fungal to bacterial ratios (Cheeke et al 2017) and are less depen-

dent on plantndashmicrobial interactions to access N than ECM trees

(Brzostek Dragoni Brown amp Phillips 2015) As such in AM-domi-

nated systems like the Fernow bacteria may outweigh fungi in con-

trolling soil decomposition responses to N fertilization Additionally

evidence that ECM trees rely more on roots and rhizosphere

microbes to access N than AM trees suggests that N fertilization

may lead to greater declines in belowground C allocation and

enzyme activity than observed at the Fernow (Brzostek et al 2015

Yin et al 2014) Moving forward the equal distribution of ECM and

AM trees across the temperate forest landscape highlights a critical

need to investigate ECM and AM stand responses to N fertilization

within the same ecosystem (Midgley amp Phillips 2014)

Although enzyme declines were observed across all three soil

fractions (Figure 1andashf) N cycling shifts were only observed in the

OH where rates were elevated by nearly 50 and 40 for mineral-

ization and nitrification respectively (Figure 4ab) OH and mineral

soils differ in key biogeochemical traits and this may contribute to

these divergent N cycling responses In temperate forests OH

0

05

1

15

Bulk Rhizo

Net

N m

iner

aliz

atio

n (

gN

middotgndash1

middotday

ndash1)

Control

+N

0

3

6

9

12

15

18

OH

0

05

1

15

Bulk Rhizo

Net

nitr

ific

atio

n ((

gN

middotgndash1

middotday

ndash1)

0

2

4

6

8

10

12

OH

(a)

(b)

Reference

F IGURE 4 N fertilization increases (a) N mineralization and (b)nitrification in the OH but not the mineral soil Values are theoverall watershed-level mean rates (mean SE) measured in JuneJuly and August 2015 for each soil fraction (n = 10 plots perwatershed) Asterisks indicate significant differences betweentreatments (p lt 05)

TABLE 2 At model steady state fertilization reduced microbialenzyme pools by ~16 and enhanced soil C by ~3

Watershed ExudationEnzyme pool(mgcm3)

Soil C pool(mgcm3)

Control No change 00136 18473

Fertilized No change 00114 19092

25 reduction 00097 22116

When root C exudation was reduced 25 on a per g root basis in the

fertilized watershed there was a further exacerbation in the reduction in

microbial enzyme activity to a ~28 decline and a larger increase in soil

C by ~20

10 | CARRARA ET AL

typically have rapid N cycling rates reflecting greater organic matter

content and root densities than mineral soils (Brzostek amp Finzi

2011) Shifts in the ratio of gross immobilization to gross mineraliza-

tion may also play a role Net N mineralization rates increased in the

OH despite N fertilization induced declines in proteolytic and chiti-

nolytic enzyme activity that produce N monomers for microbial

uptake While we do not have data on gross N cycling we hypothe-

size that declines in gross microbial N immobilization due to reduced

N demand may have outpaced declines in gross N mineralization in

the fertilized watershed Regardless of the exact mechanism it is

important to put the OH results into context on a mass per unit

area basis the OH is less important to total ecosystem N cycling

than the underlying mineral soil

Much of the research on soil N fertilization has focused on

aboveground responses such as litter input and quality or fungal

community shifts to explain widely observed reductions in enzyme

activities and subsequent soil decomposition (Edwards et al 2011

Frey et al 2004 Morrison et al 2016 Sinsabaugh et al 2008

Waldrop et al 2004 Wallenstein et al 2006) Our results provide

evidence that enzyme activity declines under long-term N fertiliza-

tion at the Fernow are driven by a cascade of ecosystem responses

whereby reductions in belowground plant C investment lead to

shifts in bacterial community structure and a decline in their ability

to degrade soil organic matter Thus there is a need to integrate

plantndashmicrobial interactions into our current conceptual and predic-

tive models of N deposition impacts on temperate forests in order

to forecast soil C responses to shifting N deposition regimes

ACKNOWLEDGEMENTS

We acknowledge Mary Beth Adams Tom Schuler and the US Forest

Service staff at the Fernow Experimental Forest for logistical assis-

tance and access to the experimental watersheds and the LTSP site

This work was also supported by the National Science Foundation

Graduate Research Fellowship to Joseph Carrara under Grant No

DGE-1102689 and by the Long-Term Research in Environmental

Biology (LTREB) program at the National Science Foundation (Grant

Nos DEB-0417678 and DEB-1019522) to William Peterjohn Colin

Averill was supported by the NOAA Climate and Global Change

Postdoctoral Fellowship Program administered by Cooperative Pro-

grams for the Advancement of Earth System Science (CPAESS)

University Corporation for Atmospheric Research (UCAR) Boulder

Colorado USA We acknowledge the WVU Genomics Core Facility

Morgantown WV for support provided to help make this publication

possible We also thank Leah Baldinger Brittany Carver Mark Burn-

ham Hannah Hedrick Jennifer Mangano and Catherine Sesa for

assistance in the field and in the laboratory

ORCID

Joseph E Carrara httporcidorg0000-0003-0597-1175

Colin Averill httporcidorg0000-0003-4035-7760

REFERENCES

Adams M B amp Angradi T R (1996) Decomposition and nutrient

dynamics of hardwood leaf litter in the Femow Whole-Watershed

Acidification Experiment Forest Ecology and Management 1127 61ndash

69 httpsdoiorg1010160378-1127(95)03695-4

Adams M B Burger J Zelazny L amp Baumgras J (2004) Descrip-

tion of the fork mountain long-term soil productivity study Site

characterization USDA Forest Service General Technical Report NE

43 323

Adams M B Edwards P J Wood F amp Kochenderfer J N (1993)

Artificial watershed acidification on the Fernow Experimental Forest

USA Journal of Hydrology 150 505ndash519 httpsdoiorg101016

0022-1694(93)90123-Q

Allison S D Wallenstein M D amp Bradford M A (2010) Soil-carbon

response to warming dependent on microbial physiology Nature Geo-

science 3 336ndash340 httpsdoiorg101038ngeo846

Bae K Fahey T J Yanai R D amp Fisk M (2015) Soil nitrogen avail-

ability affects belowground carbon allocation and soil respiration in

northern hardwood forests of new hampshire Ecosystems 18 1179ndash

1191 httpsdoiorg101007s10021-015-9892-7

Brzostek E R Dragoni D Brown Z A amp Phillips R P (2015) Mycor-

rhizal type determines the magnitude and direction of root-induced

changes in decomposition in a temperate forest New Phytologist

206 1274ndash1282 httpsdoiorg101111nph13303

Brzostek E R amp Finzi A C (2011) Substrate supply fine roots and

temperature control proteolytic enzyme activity in temperate forest

soils Ecology 92 892ndash902 httpsdoiorg10189010-18031

Brzostek E R Fisher J B amp Phillips R P (2014) Modeling the carbon

cost of plant nitrogen acquisition Mycorrhizal trade-offs and multi-

path resistance uptake improve predictions of retranslocation Journal

of Geophysical Research Biogeosciences 119 1684ndash1697

Brzostek E R Greco A Drake J E amp Finzi A C (2013) Root carbon

inputs to the rhizosphere stimulate extracellular enzyme activity and

increase nitrogen availability in temperate forest soils Biogeochem-

istry 115 65ndash76 httpsdoiorg101007s10533-012-9818-9

Burnham M B Cumming J R Adams M B amp Peterjohn W T

(2017) Soluble soil aluminum alters the relative uptake of mineral

nitrogen forms by six mature temperate broadleaf tree species

possible implications for watershed nitrate retention Oecologia

185 1ndash11

Caporaso J G Kuczynski J Stombaugh J Bittinger K Bushman F

D Costello E K Huttley G A (2010) QIIME allows analysis of

high-throughput community sequencing data Nature Methods 7

335ndash336 httpsdoiorg101038nmethf303

Carreiro M M Sinsabaugh R L Repert D A amp Parkhurst D F

(2000) Microbial enzyme shifts explain litter decay responses to sim-

ulated nitrogen deposition Ecology 81 2359ndash2365 httpsdoiorg

1018900012-9658(2000)081[2359MESELD]20CO2

Cheeke T E Phillips R P Brzostek E R Rosling A Bever J D amp

Fransson P (2017) Dominant mycorrhizal association of trees alters

carbon and nutrient cycling by selecting for microbial groups with

distinct enzyme function New Phytologist 214 432ndash442 httpsdoi

org101111nph14343

Cheng W Parton W J Gonzalez-Meler M A Phillips R Asao S

McNickle G G Jastrow J D (2014) Synthesis and modeling

perspectives of rhizosphere priming New Phytologist 201 31ndash44

httpsdoiorg101111nph12440

Comas L H Callahan H S amp Midford P E (2014) Patterns in root

traits of woody species hosting arbuscular and ectomycorrhizas

Implications for the evolution of belowground strategies Ecology and

Evolution 4 2979ndash2990 httpsdoiorg101002ece31147

Datta R Kelkar A Baraniya D Molaei A Moulick A Meena R S amp

Formanek P (2017) Enzymatic degradation of lignin in soil A

review Sustainability 9 1163 httpsdoiorg103390su9071163

CARRARA ET AL | 11

that the effects we measured are driven by N fertilization rather

than pre-existing differences between these adjacent watersheds

for four main reasons First soil chemistry (ie soil pH cation

exchange capacity nutrient content etc) were similar at the begin-

ning of the experiment (Adams amp Angradi 1996) Second the

amount of N added to the watershed yearly was originally chosen in

1989 to approximately double ambient N deposition rates but is

now more than quadruple current rates and as such it seems unli-

kely that this does not incur a biogeochemical response Third the

results from this watershed study are consistent with measurements

we made during the same year in a replicated N fertilization study

lt2 km away from these watersheds (the Fork Mountain Long-Term

Soil Productivity Plots Adams Burger Zelazny amp Baumgras 2004)

In this small-scale replicated study N fertilization reduced fine root

biomass and ligninolytic enzyme activity (Figure S5) Finally this

work builds upon other research at the Fernow that has found the

N-fertilized watershed had lower rates of litter decomposition

(Adams amp Angradi 1996) reduced understory richness (Gilliam et al

1994 2016 Walter Adams Gilliam amp Peterjohn 2017) and altered

N cycling (Adams et al 1993 Burnham Cumming Adams amp Peter-

john 2017 Gilliam Yurish amp Adams 2001 Gilliam et al 1996

2016) compared to the reference watershed

While we did observe significant declines in enzyme activity

across all three soil fractions particularly for the liginolytic enzymes

(Figure 1andashf) these declines were not correlated with significant

shifts in fungal community composition (Figure 3b) The lack of a

clear link between enzyme declines and changes in the fungal com-

munity does not support the prevailing paradigm that white-rot

Basidiomycota are the dominant cause of a decline in enzyme activi-

ties following N additions (Edwards Zak Kellner Eisenlord amp Pregit-

zer 2011 Fog 1988 Freedman Romanowicz Upchurch amp Zak

2015 Morrison et al 2016) It is possible that fungal enzyme activ-

ity response to N fertilization is independent of community composi-

tion (ie investment in enzyme activity declines with no change in

community structure) however microbial community composition

has been linked to catabolic functioning and enzyme activities across

N gradients and seasons (Fierer et al 2011 Vorıskova Brabcova

Cajthaml amp Baldrian 2014) Furthermore our data indicate that

ndash008

0

008

016

024

032

ndash09 ndash06 ndash03 0 03 06

Rhi

zo f

ungi

NM

DS2

Rhizo fungi NMDS1

ndash04

ndash02

0

02

04

ndash02 ndash01 0 01 02 03 04

Rhi

zo b

acte

ria

NM

DS2

Rhizo bacteria NMDS1

ndash02

ndash01

0

01

02

03

ndash02 0 02 04 06

Bul

k ba

cter

ia N

MD

S2

Bulk bacteria NMDS1

ndash04

ndash032

ndash024

ndash016

ndash008

0

ndash04 ndash02 0 02 04

OH

fun

gi N

MD

S2

OH fungi NMDS1

ndash06

ndash04

ndash02

0

02

ndash05 ndash04 ndash03 ndash02 ndash01 0

OH

bac

teri

a N

MD

S2

OH bacteria NMDS1

Reference

+N

ndash02

ndash01

0

01

02

03

04

ndash06 ndash04 ndash02 0 02 04

Bul

k fu

ngi N

MD

S2

Bulk fungi NMDS1

(a) (b)

(c) (d)

(e) (f)

plt01

plt01

plt01

plt01

plt01

F IGURE 2 N fertilization alteredbacterial community composition in OH (a)bulk (c) and rhizosphere (e) soils andfungal community composition in OH (b)and bulk (d) soils but not rhizosphere (f)All community data were obtained for eachsoil fraction in July 2015 (n = 10 plots perwatershed) p values indicate significantdifferences between the N-fertilized andreference community

8 | CARRARA ET AL

shifts in bacterial community composition under elevated N are cor-

related with reduced enzyme activity at the Fernow (Figure 3a)

While changes in bacterial community may be influenced by overall

fungal community composition these shifts in bacterial community

composition are tightly coupled to AM colonization (a metric of

belowground C allocation) suggesting that plant responses to N fer-

tilization feedback on bacterial community structure and function

(Figure 3c) While we cannot rule out that N-induced declines in

total fungal biomass led to reductions in liginolytic enzyme activity

(DeForest et al 2004 Frey et al 2004 Treseder 2008 Wallenstein

et al 2006) the dominance of AM trees in our plots whose inor-

ganic nutrient economy is largely driven by bacteria suggests that

free-living fungi may not be an important driver of N deposition

responses in AM-dominated systems (Cheeke et al 2017 Phillips

Brzostek amp Midgley 2013)

In contrast it appears that the declines in enzyme activity we

observed appear to be the result of a cascade of ecosystem

responses affecting both microbial community composition and plant

C allocation belowground The N-induced declines in fine root bio-

mass AM colonization and root morphology all indicate that there

was a reduction in the investment of C belowground by trees to gain

nutrients (Table 1) While previous research has shown that below-

ground C allocation is inversely correlated with N availability (Bae

Fahey Yanai amp Fisk 2015 Haynes amp Gower 1995 Phillips amp Fahey

2005) the tight linkage between the first bacterial NMDS axis and

AM colonization suggest that N-induced declines in belowground

C allocation caused a shift in the bacterial community composition

and subsequent reductions in enzyme activity (Figure 3ac) This

result also supports the emerging view that roots are active engi-

neers of microbial activity (Brzostek et al 2013 Finzi et al 2015

Yin et al 2014) and highlights the need to integrate root-microbial

interactions into our conceptual and predictive modeling frameworks

of how forest ecosystems respond to global change (Johnson

Gehring amp Jansa 2016 Terrer Vicca Hungate Phillips amp Prentice

2016)

An additional impact of the N fertilization treatment is that it

reduced soil pH (as measured in a 001 M calcium chloride buffer) of

the upper 5 cm of mineral soil in the fertilized watershed to 34

compared to 38 in the control watershed (Peterjohn unpublished

data) Soil pH is an important control on microbial community diver-

sity (Fierer et al 2012 Kaiser et al 2016 Lauber Hamady Knight

amp Fierer 2009) microbial biomass and enzyme activities (Rousk amp

Baath 2011 Sinsabaugh 2010) Thus the decline in soil pH may

account for a portion of the enzyme reductions we observed under

N fertilization To address this we assayed the sensitivity of phenol

oxidase and peroxidase enzyme activity in organic horizon bulk and

rhizosphere soils from the control watershed to three different levels

of pH that spanned a 1 unit shift (data not shown) Peroxidase activ-

ity was insensitive to pH (slope = 074 r2 = 05 p gt 05) While

phenol oxidase activity did significantly increase as pH increased

(slope = 010 r2 = 053 p lt 05) this sensitivity would only account

for a 10 decline in activity Given that we observed a 50 decline

in phenol oxidase in the treatment watershed coupled with the

greater overall importance of peroxidase enzymes in our study (ie

nearly an order of magnitude higher activity) it appears that pH is

an important but secondary driver of the enzyme activity responses

we observed

Over longer time scales the reductions in root and microbial

activity we observed at the Fernow may have important implications

for soil C stocks In our model simulation we found that feedbacks

between reductions in root biomass and enzyme production have

the potential to drive nearly a 3 increase in soil C stocks (Table 2)

When these were coupled with a 25 reduction in specific root C

exudation rates soil C in the fertilized watershed increased by nearly

20 over the 30-yr simulation This model was designed to be theo-

retical and as such it is used here to show the sensitivity of the

y = 0004x ndash 007R = 63

ndash02

0

02

04

06

0 25 50 75 100Bul

k B

acte

ria

OT

U N

MD

S1

AM Colonization ()

y = ndash0159x + 013R = 360

004

008

012

016

02

ndash02 0 02 04 06

Enz

yme

NM

DS

1

Bacteria OTU NMDS1

+NReference

0

004

008

012

016

02

ndash06 ndash04 ndash02 0 02 04

Enz

yme

NM

DS

1

Fungal OTU NMDS1

ndash06

ndash04

ndash02

0

02

04

0 25 50 75 100

Bul

k Fu

ngi

OT

U N

MD

S1

AM Colonization ()

plt05

plt001

(a)

(c)

(b)

(d)F IGURE 3 Bacterial but not fungalcommunity composition is correlated withthe first NMDS axis of enzyme activity (aamp b) Percent AM colonization is correlatedwith bacterial community composition (c)but not fungal community composition (d)Data presented are bulk soil communitycomposition data from July 2015 (n = 10plots per watershed) p values indicatesignificance of correlation

CARRARA ET AL | 9

system to the perturbations in plant inputs and is not meant to be

quantitatively predictive However this simple modeling effort pro-

vides compelling evidence that N-induced shifts in rootndashmicrobial

interactions have the potential to alter not only microbial production

of soil enzymes but also the size of the soil C pool To generate the

necessary data for fully integrating root-microbial interactions into

more sophisticated ecosystem models future research should couple

direct measurements of belowground C allocation responses to N

fertilization (eg total belowground C allocation root exudation)

with the resulting impacts on microbial activity

Given the prevailing paradigm that free-living fungi drive soil

enzyme activity and subsequent decomposition responses to N fer-

tilization (Carreiro et al 2000 Fog 1988 Waldrop et al 2004 Zak

et al 2008) our results raise an important question of why shifts in

plant C allocation bacterial community structure and enzyme activi-

ties were tightly coupled at the Fernow The divergent results

between the Fernow and other long-term fertilization sites may be

the result of differences in ambient N deposition and tree species

composition The Fernow is in a region that has historically received

some of the highest levels of N deposition in the United States

(Driscoll et al 2001) which may have enhanced bacterial dominance

before treatment began in 1989 By contrast N fertilization experi-

ments that have linked reductions in soil C decomposition to fungal

community composition and activity tend to be located in areas with

lower historical N deposition rates (Edwards et al 2011 Freedman

et al 2015 Frey et al 2004 Morrison et al 2016 Wallenstein

et al 2006) Second the Fernow is predominantly dominated by

AM trees whereas most N fertilization experiments have tended to

occur in ecosystems dominated by ECM trees with ECM-dominated

sites comprising ~80 of the studies included in the Janssens et al

(2010) meta-analysis AM trees show greater growth enhancements

with N addition (Thomas et al 2010) are characterized by lower

fungal to bacterial ratios (Cheeke et al 2017) and are less depen-

dent on plantndashmicrobial interactions to access N than ECM trees

(Brzostek Dragoni Brown amp Phillips 2015) As such in AM-domi-

nated systems like the Fernow bacteria may outweigh fungi in con-

trolling soil decomposition responses to N fertilization Additionally

evidence that ECM trees rely more on roots and rhizosphere

microbes to access N than AM trees suggests that N fertilization

may lead to greater declines in belowground C allocation and

enzyme activity than observed at the Fernow (Brzostek et al 2015

Yin et al 2014) Moving forward the equal distribution of ECM and

AM trees across the temperate forest landscape highlights a critical

need to investigate ECM and AM stand responses to N fertilization

within the same ecosystem (Midgley amp Phillips 2014)

Although enzyme declines were observed across all three soil

fractions (Figure 1andashf) N cycling shifts were only observed in the

OH where rates were elevated by nearly 50 and 40 for mineral-

ization and nitrification respectively (Figure 4ab) OH and mineral

soils differ in key biogeochemical traits and this may contribute to

these divergent N cycling responses In temperate forests OH

0

05

1

15

Bulk Rhizo

Net

N m

iner

aliz

atio

n (

gN

middotgndash1

middotday

ndash1)

Control

+N

0

3

6

9

12

15

18

OH

0

05

1

15

Bulk Rhizo

Net

nitr

ific

atio

n ((

gN

middotgndash1

middotday

ndash1)

0

2

4

6

8

10

12

OH

(a)

(b)

Reference

F IGURE 4 N fertilization increases (a) N mineralization and (b)nitrification in the OH but not the mineral soil Values are theoverall watershed-level mean rates (mean SE) measured in JuneJuly and August 2015 for each soil fraction (n = 10 plots perwatershed) Asterisks indicate significant differences betweentreatments (p lt 05)

TABLE 2 At model steady state fertilization reduced microbialenzyme pools by ~16 and enhanced soil C by ~3

Watershed ExudationEnzyme pool(mgcm3)

Soil C pool(mgcm3)

Control No change 00136 18473

Fertilized No change 00114 19092

25 reduction 00097 22116

When root C exudation was reduced 25 on a per g root basis in the

fertilized watershed there was a further exacerbation in the reduction in

microbial enzyme activity to a ~28 decline and a larger increase in soil

C by ~20

10 | CARRARA ET AL

typically have rapid N cycling rates reflecting greater organic matter

content and root densities than mineral soils (Brzostek amp Finzi

2011) Shifts in the ratio of gross immobilization to gross mineraliza-

tion may also play a role Net N mineralization rates increased in the

OH despite N fertilization induced declines in proteolytic and chiti-

nolytic enzyme activity that produce N monomers for microbial

uptake While we do not have data on gross N cycling we hypothe-

size that declines in gross microbial N immobilization due to reduced

N demand may have outpaced declines in gross N mineralization in

the fertilized watershed Regardless of the exact mechanism it is

important to put the OH results into context on a mass per unit

area basis the OH is less important to total ecosystem N cycling

than the underlying mineral soil

Much of the research on soil N fertilization has focused on

aboveground responses such as litter input and quality or fungal

community shifts to explain widely observed reductions in enzyme

activities and subsequent soil decomposition (Edwards et al 2011

Frey et al 2004 Morrison et al 2016 Sinsabaugh et al 2008

Waldrop et al 2004 Wallenstein et al 2006) Our results provide

evidence that enzyme activity declines under long-term N fertiliza-

tion at the Fernow are driven by a cascade of ecosystem responses

whereby reductions in belowground plant C investment lead to

shifts in bacterial community structure and a decline in their ability

to degrade soil organic matter Thus there is a need to integrate

plantndashmicrobial interactions into our current conceptual and predic-

tive models of N deposition impacts on temperate forests in order

to forecast soil C responses to shifting N deposition regimes

ACKNOWLEDGEMENTS

We acknowledge Mary Beth Adams Tom Schuler and the US Forest

Service staff at the Fernow Experimental Forest for logistical assis-

tance and access to the experimental watersheds and the LTSP site

This work was also supported by the National Science Foundation

Graduate Research Fellowship to Joseph Carrara under Grant No

DGE-1102689 and by the Long-Term Research in Environmental

Biology (LTREB) program at the National Science Foundation (Grant

Nos DEB-0417678 and DEB-1019522) to William Peterjohn Colin

Averill was supported by the NOAA Climate and Global Change

Postdoctoral Fellowship Program administered by Cooperative Pro-

grams for the Advancement of Earth System Science (CPAESS)

University Corporation for Atmospheric Research (UCAR) Boulder

Colorado USA We acknowledge the WVU Genomics Core Facility

Morgantown WV for support provided to help make this publication

possible We also thank Leah Baldinger Brittany Carver Mark Burn-

ham Hannah Hedrick Jennifer Mangano and Catherine Sesa for

assistance in the field and in the laboratory

ORCID

Joseph E Carrara httporcidorg0000-0003-0597-1175

Colin Averill httporcidorg0000-0003-4035-7760

REFERENCES

Adams M B amp Angradi T R (1996) Decomposition and nutrient

dynamics of hardwood leaf litter in the Femow Whole-Watershed

Acidification Experiment Forest Ecology and Management 1127 61ndash

69 httpsdoiorg1010160378-1127(95)03695-4

Adams M B Burger J Zelazny L amp Baumgras J (2004) Descrip-

tion of the fork mountain long-term soil productivity study Site

characterization USDA Forest Service General Technical Report NE

43 323

Adams M B Edwards P J Wood F amp Kochenderfer J N (1993)

Artificial watershed acidification on the Fernow Experimental Forest

USA Journal of Hydrology 150 505ndash519 httpsdoiorg101016

0022-1694(93)90123-Q

Allison S D Wallenstein M D amp Bradford M A (2010) Soil-carbon

response to warming dependent on microbial physiology Nature Geo-

science 3 336ndash340 httpsdoiorg101038ngeo846

Bae K Fahey T J Yanai R D amp Fisk M (2015) Soil nitrogen avail-

ability affects belowground carbon allocation and soil respiration in

northern hardwood forests of new hampshire Ecosystems 18 1179ndash

1191 httpsdoiorg101007s10021-015-9892-7

Brzostek E R Dragoni D Brown Z A amp Phillips R P (2015) Mycor-

rhizal type determines the magnitude and direction of root-induced

changes in decomposition in a temperate forest New Phytologist

206 1274ndash1282 httpsdoiorg101111nph13303

Brzostek E R amp Finzi A C (2011) Substrate supply fine roots and

temperature control proteolytic enzyme activity in temperate forest

soils Ecology 92 892ndash902 httpsdoiorg10189010-18031

Brzostek E R Fisher J B amp Phillips R P (2014) Modeling the carbon

cost of plant nitrogen acquisition Mycorrhizal trade-offs and multi-

path resistance uptake improve predictions of retranslocation Journal

of Geophysical Research Biogeosciences 119 1684ndash1697

Brzostek E R Greco A Drake J E amp Finzi A C (2013) Root carbon

inputs to the rhizosphere stimulate extracellular enzyme activity and

increase nitrogen availability in temperate forest soils Biogeochem-

istry 115 65ndash76 httpsdoiorg101007s10533-012-9818-9

Burnham M B Cumming J R Adams M B amp Peterjohn W T

(2017) Soluble soil aluminum alters the relative uptake of mineral

nitrogen forms by six mature temperate broadleaf tree species

possible implications for watershed nitrate retention Oecologia

185 1ndash11

Caporaso J G Kuczynski J Stombaugh J Bittinger K Bushman F

D Costello E K Huttley G A (2010) QIIME allows analysis of

high-throughput community sequencing data Nature Methods 7

335ndash336 httpsdoiorg101038nmethf303

Carreiro M M Sinsabaugh R L Repert D A amp Parkhurst D F

(2000) Microbial enzyme shifts explain litter decay responses to sim-

ulated nitrogen deposition Ecology 81 2359ndash2365 httpsdoiorg

1018900012-9658(2000)081[2359MESELD]20CO2

Cheeke T E Phillips R P Brzostek E R Rosling A Bever J D amp

Fransson P (2017) Dominant mycorrhizal association of trees alters

carbon and nutrient cycling by selecting for microbial groups with

distinct enzyme function New Phytologist 214 432ndash442 httpsdoi

org101111nph14343

Cheng W Parton W J Gonzalez-Meler M A Phillips R Asao S

McNickle G G Jastrow J D (2014) Synthesis and modeling

perspectives of rhizosphere priming New Phytologist 201 31ndash44

httpsdoiorg101111nph12440

Comas L H Callahan H S amp Midford P E (2014) Patterns in root

traits of woody species hosting arbuscular and ectomycorrhizas

Implications for the evolution of belowground strategies Ecology and

Evolution 4 2979ndash2990 httpsdoiorg101002ece31147

Datta R Kelkar A Baraniya D Molaei A Moulick A Meena R S amp

Formanek P (2017) Enzymatic degradation of lignin in soil A

review Sustainability 9 1163 httpsdoiorg103390su9071163

CARRARA ET AL | 11

shifts in bacterial community composition under elevated N are cor-

related with reduced enzyme activity at the Fernow (Figure 3a)

While changes in bacterial community may be influenced by overall

fungal community composition these shifts in bacterial community

composition are tightly coupled to AM colonization (a metric of

belowground C allocation) suggesting that plant responses to N fer-

tilization feedback on bacterial community structure and function

(Figure 3c) While we cannot rule out that N-induced declines in

total fungal biomass led to reductions in liginolytic enzyme activity

(DeForest et al 2004 Frey et al 2004 Treseder 2008 Wallenstein

et al 2006) the dominance of AM trees in our plots whose inor-

ganic nutrient economy is largely driven by bacteria suggests that

free-living fungi may not be an important driver of N deposition

responses in AM-dominated systems (Cheeke et al 2017 Phillips

Brzostek amp Midgley 2013)

In contrast it appears that the declines in enzyme activity we

observed appear to be the result of a cascade of ecosystem

responses affecting both microbial community composition and plant

C allocation belowground The N-induced declines in fine root bio-

mass AM colonization and root morphology all indicate that there

was a reduction in the investment of C belowground by trees to gain

nutrients (Table 1) While previous research has shown that below-

ground C allocation is inversely correlated with N availability (Bae

Fahey Yanai amp Fisk 2015 Haynes amp Gower 1995 Phillips amp Fahey

2005) the tight linkage between the first bacterial NMDS axis and

AM colonization suggest that N-induced declines in belowground

C allocation caused a shift in the bacterial community composition

and subsequent reductions in enzyme activity (Figure 3ac) This

result also supports the emerging view that roots are active engi-

neers of microbial activity (Brzostek et al 2013 Finzi et al 2015

Yin et al 2014) and highlights the need to integrate root-microbial

interactions into our conceptual and predictive modeling frameworks

of how forest ecosystems respond to global change (Johnson

Gehring amp Jansa 2016 Terrer Vicca Hungate Phillips amp Prentice

2016)

An additional impact of the N fertilization treatment is that it

reduced soil pH (as measured in a 001 M calcium chloride buffer) of

the upper 5 cm of mineral soil in the fertilized watershed to 34

compared to 38 in the control watershed (Peterjohn unpublished

data) Soil pH is an important control on microbial community diver-

sity (Fierer et al 2012 Kaiser et al 2016 Lauber Hamady Knight

amp Fierer 2009) microbial biomass and enzyme activities (Rousk amp

Baath 2011 Sinsabaugh 2010) Thus the decline in soil pH may

account for a portion of the enzyme reductions we observed under

N fertilization To address this we assayed the sensitivity of phenol

oxidase and peroxidase enzyme activity in organic horizon bulk and

rhizosphere soils from the control watershed to three different levels

of pH that spanned a 1 unit shift (data not shown) Peroxidase activ-

ity was insensitive to pH (slope = 074 r2 = 05 p gt 05) While

phenol oxidase activity did significantly increase as pH increased

(slope = 010 r2 = 053 p lt 05) this sensitivity would only account

for a 10 decline in activity Given that we observed a 50 decline

in phenol oxidase in the treatment watershed coupled with the

greater overall importance of peroxidase enzymes in our study (ie

nearly an order of magnitude higher activity) it appears that pH is

an important but secondary driver of the enzyme activity responses

we observed

Over longer time scales the reductions in root and microbial

activity we observed at the Fernow may have important implications

for soil C stocks In our model simulation we found that feedbacks

between reductions in root biomass and enzyme production have

the potential to drive nearly a 3 increase in soil C stocks (Table 2)

When these were coupled with a 25 reduction in specific root C

exudation rates soil C in the fertilized watershed increased by nearly

20 over the 30-yr simulation This model was designed to be theo-

retical and as such it is used here to show the sensitivity of the

y = 0004x ndash 007R = 63

ndash02

0

02

04

06

0 25 50 75 100Bul

k B

acte

ria

OT

U N

MD

S1

AM Colonization ()

y = ndash0159x + 013R = 360

004

008

012

016

02

ndash02 0 02 04 06

Enz

yme

NM

DS

1

Bacteria OTU NMDS1

+NReference

0

004

008

012

016

02

ndash06 ndash04 ndash02 0 02 04

Enz

yme

NM

DS

1

Fungal OTU NMDS1

ndash06

ndash04

ndash02

0

02

04

0 25 50 75 100

Bul

k Fu

ngi

OT

U N

MD

S1

AM Colonization ()

plt05

plt001

(a)

(c)

(b)

(d)F IGURE 3 Bacterial but not fungalcommunity composition is correlated withthe first NMDS axis of enzyme activity (aamp b) Percent AM colonization is correlatedwith bacterial community composition (c)but not fungal community composition (d)Data presented are bulk soil communitycomposition data from July 2015 (n = 10plots per watershed) p values indicatesignificance of correlation

CARRARA ET AL | 9

system to the perturbations in plant inputs and is not meant to be

quantitatively predictive However this simple modeling effort pro-

vides compelling evidence that N-induced shifts in rootndashmicrobial

interactions have the potential to alter not only microbial production

of soil enzymes but also the size of the soil C pool To generate the

necessary data for fully integrating root-microbial interactions into

more sophisticated ecosystem models future research should couple

direct measurements of belowground C allocation responses to N

fertilization (eg total belowground C allocation root exudation)

with the resulting impacts on microbial activity

Given the prevailing paradigm that free-living fungi drive soil

enzyme activity and subsequent decomposition responses to N fer-

tilization (Carreiro et al 2000 Fog 1988 Waldrop et al 2004 Zak

et al 2008) our results raise an important question of why shifts in

plant C allocation bacterial community structure and enzyme activi-

ties were tightly coupled at the Fernow The divergent results

between the Fernow and other long-term fertilization sites may be

the result of differences in ambient N deposition and tree species

composition The Fernow is in a region that has historically received

some of the highest levels of N deposition in the United States

(Driscoll et al 2001) which may have enhanced bacterial dominance

before treatment began in 1989 By contrast N fertilization experi-

ments that have linked reductions in soil C decomposition to fungal

community composition and activity tend to be located in areas with

lower historical N deposition rates (Edwards et al 2011 Freedman

et al 2015 Frey et al 2004 Morrison et al 2016 Wallenstein

et al 2006) Second the Fernow is predominantly dominated by

AM trees whereas most N fertilization experiments have tended to

occur in ecosystems dominated by ECM trees with ECM-dominated

sites comprising ~80 of the studies included in the Janssens et al

(2010) meta-analysis AM trees show greater growth enhancements

with N addition (Thomas et al 2010) are characterized by lower

fungal to bacterial ratios (Cheeke et al 2017) and are less depen-

dent on plantndashmicrobial interactions to access N than ECM trees

(Brzostek Dragoni Brown amp Phillips 2015) As such in AM-domi-

nated systems like the Fernow bacteria may outweigh fungi in con-

trolling soil decomposition responses to N fertilization Additionally

evidence that ECM trees rely more on roots and rhizosphere

microbes to access N than AM trees suggests that N fertilization

may lead to greater declines in belowground C allocation and

enzyme activity than observed at the Fernow (Brzostek et al 2015

Yin et al 2014) Moving forward the equal distribution of ECM and

AM trees across the temperate forest landscape highlights a critical

need to investigate ECM and AM stand responses to N fertilization

within the same ecosystem (Midgley amp Phillips 2014)

Although enzyme declines were observed across all three soil

fractions (Figure 1andashf) N cycling shifts were only observed in the

OH where rates were elevated by nearly 50 and 40 for mineral-

ization and nitrification respectively (Figure 4ab) OH and mineral

soils differ in key biogeochemical traits and this may contribute to

these divergent N cycling responses In temperate forests OH

0

05

1

15

Bulk Rhizo

Net

N m

iner

aliz

atio

n (

gN

middotgndash1

middotday

ndash1)

Control

+N

0

3

6

9

12

15

18

OH

0

05

1

15

Bulk Rhizo

Net

nitr

ific

atio

n ((

gN

middotgndash1

middotday

ndash1)

0

2

4

6

8

10

12

OH

(a)

(b)

Reference

F IGURE 4 N fertilization increases (a) N mineralization and (b)nitrification in the OH but not the mineral soil Values are theoverall watershed-level mean rates (mean SE) measured in JuneJuly and August 2015 for each soil fraction (n = 10 plots perwatershed) Asterisks indicate significant differences betweentreatments (p lt 05)

TABLE 2 At model steady state fertilization reduced microbialenzyme pools by ~16 and enhanced soil C by ~3

Watershed ExudationEnzyme pool(mgcm3)

Soil C pool(mgcm3)

Control No change 00136 18473

Fertilized No change 00114 19092

25 reduction 00097 22116

When root C exudation was reduced 25 on a per g root basis in the

fertilized watershed there was a further exacerbation in the reduction in

microbial enzyme activity to a ~28 decline and a larger increase in soil

C by ~20

10 | CARRARA ET AL

typically have rapid N cycling rates reflecting greater organic matter

content and root densities than mineral soils (Brzostek amp Finzi

2011) Shifts in the ratio of gross immobilization to gross mineraliza-

tion may also play a role Net N mineralization rates increased in the

OH despite N fertilization induced declines in proteolytic and chiti-

nolytic enzyme activity that produce N monomers for microbial

uptake While we do not have data on gross N cycling we hypothe-

size that declines in gross microbial N immobilization due to reduced

N demand may have outpaced declines in gross N mineralization in

the fertilized watershed Regardless of the exact mechanism it is

important to put the OH results into context on a mass per unit

area basis the OH is less important to total ecosystem N cycling

than the underlying mineral soil

Much of the research on soil N fertilization has focused on

aboveground responses such as litter input and quality or fungal

community shifts to explain widely observed reductions in enzyme

activities and subsequent soil decomposition (Edwards et al 2011

Frey et al 2004 Morrison et al 2016 Sinsabaugh et al 2008

Waldrop et al 2004 Wallenstein et al 2006) Our results provide

evidence that enzyme activity declines under long-term N fertiliza-

tion at the Fernow are driven by a cascade of ecosystem responses

whereby reductions in belowground plant C investment lead to

shifts in bacterial community structure and a decline in their ability

to degrade soil organic matter Thus there is a need to integrate

plantndashmicrobial interactions into our current conceptual and predic-

tive models of N deposition impacts on temperate forests in order

to forecast soil C responses to shifting N deposition regimes

ACKNOWLEDGEMENTS

We acknowledge Mary Beth Adams Tom Schuler and the US Forest

Service staff at the Fernow Experimental Forest for logistical assis-

tance and access to the experimental watersheds and the LTSP site

This work was also supported by the National Science Foundation

Graduate Research Fellowship to Joseph Carrara under Grant No

DGE-1102689 and by the Long-Term Research in Environmental

Biology (LTREB) program at the National Science Foundation (Grant

Nos DEB-0417678 and DEB-1019522) to William Peterjohn Colin

Averill was supported by the NOAA Climate and Global Change

Postdoctoral Fellowship Program administered by Cooperative Pro-

grams for the Advancement of Earth System Science (CPAESS)

University Corporation for Atmospheric Research (UCAR) Boulder

Colorado USA We acknowledge the WVU Genomics Core Facility

Morgantown WV for support provided to help make this publication

possible We also thank Leah Baldinger Brittany Carver Mark Burn-

ham Hannah Hedrick Jennifer Mangano and Catherine Sesa for

assistance in the field and in the laboratory

ORCID

Joseph E Carrara httporcidorg0000-0003-0597-1175

Colin Averill httporcidorg0000-0003-4035-7760

REFERENCES

Adams M B amp Angradi T R (1996) Decomposition and nutrient

dynamics of hardwood leaf litter in the Femow Whole-Watershed

Acidification Experiment Forest Ecology and Management 1127 61ndash

69 httpsdoiorg1010160378-1127(95)03695-4

Adams M B Burger J Zelazny L amp Baumgras J (2004) Descrip-

tion of the fork mountain long-term soil productivity study Site

characterization USDA Forest Service General Technical Report NE

43 323

Adams M B Edwards P J Wood F amp Kochenderfer J N (1993)

Artificial watershed acidification on the Fernow Experimental Forest

USA Journal of Hydrology 150 505ndash519 httpsdoiorg101016

0022-1694(93)90123-Q

Allison S D Wallenstein M D amp Bradford M A (2010) Soil-carbon

response to warming dependent on microbial physiology Nature Geo-

science 3 336ndash340 httpsdoiorg101038ngeo846

Bae K Fahey T J Yanai R D amp Fisk M (2015) Soil nitrogen avail-

ability affects belowground carbon allocation and soil respiration in

northern hardwood forests of new hampshire Ecosystems 18 1179ndash

1191 httpsdoiorg101007s10021-015-9892-7

Brzostek E R Dragoni D Brown Z A amp Phillips R P (2015) Mycor-

rhizal type determines the magnitude and direction of root-induced

changes in decomposition in a temperate forest New Phytologist

206 1274ndash1282 httpsdoiorg101111nph13303

Brzostek E R amp Finzi A C (2011) Substrate supply fine roots and

temperature control proteolytic enzyme activity in temperate forest

soils Ecology 92 892ndash902 httpsdoiorg10189010-18031

Brzostek E R Fisher J B amp Phillips R P (2014) Modeling the carbon

cost of plant nitrogen acquisition Mycorrhizal trade-offs and multi-

path resistance uptake improve predictions of retranslocation Journal

of Geophysical Research Biogeosciences 119 1684ndash1697

Brzostek E R Greco A Drake J E amp Finzi A C (2013) Root carbon

inputs to the rhizosphere stimulate extracellular enzyme activity and

increase nitrogen availability in temperate forest soils Biogeochem-

istry 115 65ndash76 httpsdoiorg101007s10533-012-9818-9

Burnham M B Cumming J R Adams M B amp Peterjohn W T

(2017) Soluble soil aluminum alters the relative uptake of mineral

nitrogen forms by six mature temperate broadleaf tree species

possible implications for watershed nitrate retention Oecologia

185 1ndash11

Caporaso J G Kuczynski J Stombaugh J Bittinger K Bushman F

D Costello E K Huttley G A (2010) QIIME allows analysis of

high-throughput community sequencing data Nature Methods 7

335ndash336 httpsdoiorg101038nmethf303

Carreiro M M Sinsabaugh R L Repert D A amp Parkhurst D F

(2000) Microbial enzyme shifts explain litter decay responses to sim-

ulated nitrogen deposition Ecology 81 2359ndash2365 httpsdoiorg

1018900012-9658(2000)081[2359MESELD]20CO2

Cheeke T E Phillips R P Brzostek E R Rosling A Bever J D amp

Fransson P (2017) Dominant mycorrhizal association of trees alters

carbon and nutrient cycling by selecting for microbial groups with

distinct enzyme function New Phytologist 214 432ndash442 httpsdoi

org101111nph14343

Cheng W Parton W J Gonzalez-Meler M A Phillips R Asao S

McNickle G G Jastrow J D (2014) Synthesis and modeling

perspectives of rhizosphere priming New Phytologist 201 31ndash44

httpsdoiorg101111nph12440

Comas L H Callahan H S amp Midford P E (2014) Patterns in root

traits of woody species hosting arbuscular and ectomycorrhizas

Implications for the evolution of belowground strategies Ecology and

Evolution 4 2979ndash2990 httpsdoiorg101002ece31147

Datta R Kelkar A Baraniya D Molaei A Moulick A Meena R S amp

Formanek P (2017) Enzymatic degradation of lignin in soil A

review Sustainability 9 1163 httpsdoiorg103390su9071163

CARRARA ET AL | 11

system to the perturbations in plant inputs and is not meant to be

quantitatively predictive However this simple modeling effort pro-

vides compelling evidence that N-induced shifts in rootndashmicrobial

interactions have the potential to alter not only microbial production

of soil enzymes but also the size of the soil C pool To generate the

necessary data for fully integrating root-microbial interactions into

more sophisticated ecosystem models future research should couple

direct measurements of belowground C allocation responses to N

fertilization (eg total belowground C allocation root exudation)

with the resulting impacts on microbial activity

Given the prevailing paradigm that free-living fungi drive soil

enzyme activity and subsequent decomposition responses to N fer-

tilization (Carreiro et al 2000 Fog 1988 Waldrop et al 2004 Zak

et al 2008) our results raise an important question of why shifts in

plant C allocation bacterial community structure and enzyme activi-

ties were tightly coupled at the Fernow The divergent results

between the Fernow and other long-term fertilization sites may be

the result of differences in ambient N deposition and tree species

composition The Fernow is in a region that has historically received

some of the highest levels of N deposition in the United States

(Driscoll et al 2001) which may have enhanced bacterial dominance

before treatment began in 1989 By contrast N fertilization experi-

ments that have linked reductions in soil C decomposition to fungal

community composition and activity tend to be located in areas with

lower historical N deposition rates (Edwards et al 2011 Freedman

et al 2015 Frey et al 2004 Morrison et al 2016 Wallenstein

et al 2006) Second the Fernow is predominantly dominated by

AM trees whereas most N fertilization experiments have tended to

occur in ecosystems dominated by ECM trees with ECM-dominated

sites comprising ~80 of the studies included in the Janssens et al

(2010) meta-analysis AM trees show greater growth enhancements

with N addition (Thomas et al 2010) are characterized by lower

fungal to bacterial ratios (Cheeke et al 2017) and are less depen-

dent on plantndashmicrobial interactions to access N than ECM trees

(Brzostek Dragoni Brown amp Phillips 2015) As such in AM-domi-

nated systems like the Fernow bacteria may outweigh fungi in con-

trolling soil decomposition responses to N fertilization Additionally

evidence that ECM trees rely more on roots and rhizosphere

microbes to access N than AM trees suggests that N fertilization

may lead to greater declines in belowground C allocation and

enzyme activity than observed at the Fernow (Brzostek et al 2015

Yin et al 2014) Moving forward the equal distribution of ECM and

AM trees across the temperate forest landscape highlights a critical

need to investigate ECM and AM stand responses to N fertilization

within the same ecosystem (Midgley amp Phillips 2014)

Although enzyme declines were observed across all three soil

fractions (Figure 1andashf) N cycling shifts were only observed in the

OH where rates were elevated by nearly 50 and 40 for mineral-

ization and nitrification respectively (Figure 4ab) OH and mineral

soils differ in key biogeochemical traits and this may contribute to

these divergent N cycling responses In temperate forests OH

0

05

1

15

Bulk Rhizo

Net

N m

iner

aliz

atio

n (

gN

middotgndash1

middotday

ndash1)

Control

+N

0

3

6

9

12

15

18

OH

0

05

1

15

Bulk Rhizo

Net

nitr

ific

atio

n ((

gN

middotgndash1

middotday

ndash1)

0

2

4

6

8

10

12

OH

(a)

(b)

Reference

F IGURE 4 N fertilization increases (a) N mineralization and (b)nitrification in the OH but not the mineral soil Values are theoverall watershed-level mean rates (mean SE) measured in JuneJuly and August 2015 for each soil fraction (n = 10 plots perwatershed) Asterisks indicate significant differences betweentreatments (p lt 05)

TABLE 2 At model steady state fertilization reduced microbialenzyme pools by ~16 and enhanced soil C by ~3

Watershed ExudationEnzyme pool(mgcm3)

Soil C pool(mgcm3)

Control No change 00136 18473

Fertilized No change 00114 19092

25 reduction 00097 22116

When root C exudation was reduced 25 on a per g root basis in the

fertilized watershed there was a further exacerbation in the reduction in

microbial enzyme activity to a ~28 decline and a larger increase in soil

C by ~20

10 | CARRARA ET AL

typically have rapid N cycling rates reflecting greater organic matter

content and root densities than mineral soils (Brzostek amp Finzi

2011) Shifts in the ratio of gross immobilization to gross mineraliza-

tion may also play a role Net N mineralization rates increased in the

OH despite N fertilization induced declines in proteolytic and chiti-

nolytic enzyme activity that produce N monomers for microbial

uptake While we do not have data on gross N cycling we hypothe-

size that declines in gross microbial N immobilization due to reduced

N demand may have outpaced declines in gross N mineralization in

the fertilized watershed Regardless of the exact mechanism it is

important to put the OH results into context on a mass per unit

area basis the OH is less important to total ecosystem N cycling

than the underlying mineral soil

Much of the research on soil N fertilization has focused on

aboveground responses such as litter input and quality or fungal

community shifts to explain widely observed reductions in enzyme

activities and subsequent soil decomposition (Edwards et al 2011

Frey et al 2004 Morrison et al 2016 Sinsabaugh et al 2008

Waldrop et al 2004 Wallenstein et al 2006) Our results provide

evidence that enzyme activity declines under long-term N fertiliza-

tion at the Fernow are driven by a cascade of ecosystem responses

whereby reductions in belowground plant C investment lead to

shifts in bacterial community structure and a decline in their ability

to degrade soil organic matter Thus there is a need to integrate

plantndashmicrobial interactions into our current conceptual and predic-

tive models of N deposition impacts on temperate forests in order

to forecast soil C responses to shifting N deposition regimes

ACKNOWLEDGEMENTS

We acknowledge Mary Beth Adams Tom Schuler and the US Forest

Service staff at the Fernow Experimental Forest for logistical assis-

tance and access to the experimental watersheds and the LTSP site

This work was also supported by the National Science Foundation

Graduate Research Fellowship to Joseph Carrara under Grant No

DGE-1102689 and by the Long-Term Research in Environmental

Biology (LTREB) program at the National Science Foundation (Grant

Nos DEB-0417678 and DEB-1019522) to William Peterjohn Colin

Averill was supported by the NOAA Climate and Global Change

Postdoctoral Fellowship Program administered by Cooperative Pro-

grams for the Advancement of Earth System Science (CPAESS)

University Corporation for Atmospheric Research (UCAR) Boulder

Colorado USA We acknowledge the WVU Genomics Core Facility

Morgantown WV for support provided to help make this publication

possible We also thank Leah Baldinger Brittany Carver Mark Burn-

ham Hannah Hedrick Jennifer Mangano and Catherine Sesa for

assistance in the field and in the laboratory

ORCID

Joseph E Carrara httporcidorg0000-0003-0597-1175

Colin Averill httporcidorg0000-0003-4035-7760

REFERENCES

Adams M B amp Angradi T R (1996) Decomposition and nutrient

dynamics of hardwood leaf litter in the Femow Whole-Watershed

Acidification Experiment Forest Ecology and Management 1127 61ndash

69 httpsdoiorg1010160378-1127(95)03695-4

Adams M B Burger J Zelazny L amp Baumgras J (2004) Descrip-

tion of the fork mountain long-term soil productivity study Site

characterization USDA Forest Service General Technical Report NE

43 323

Adams M B Edwards P J Wood F amp Kochenderfer J N (1993)

Artificial watershed acidification on the Fernow Experimental Forest

USA Journal of Hydrology 150 505ndash519 httpsdoiorg101016

0022-1694(93)90123-Q

Allison S D Wallenstein M D amp Bradford M A (2010) Soil-carbon

response to warming dependent on microbial physiology Nature Geo-

science 3 336ndash340 httpsdoiorg101038ngeo846

Bae K Fahey T J Yanai R D amp Fisk M (2015) Soil nitrogen avail-

ability affects belowground carbon allocation and soil respiration in

northern hardwood forests of new hampshire Ecosystems 18 1179ndash

1191 httpsdoiorg101007s10021-015-9892-7

Brzostek E R Dragoni D Brown Z A amp Phillips R P (2015) Mycor-

rhizal type determines the magnitude and direction of root-induced

changes in decomposition in a temperate forest New Phytologist

206 1274ndash1282 httpsdoiorg101111nph13303

Brzostek E R amp Finzi A C (2011) Substrate supply fine roots and

temperature control proteolytic enzyme activity in temperate forest

soils Ecology 92 892ndash902 httpsdoiorg10189010-18031

Brzostek E R Fisher J B amp Phillips R P (2014) Modeling the carbon

cost of plant nitrogen acquisition Mycorrhizal trade-offs and multi-

path resistance uptake improve predictions of retranslocation Journal

of Geophysical Research Biogeosciences 119 1684ndash1697

Brzostek E R Greco A Drake J E amp Finzi A C (2013) Root carbon

inputs to the rhizosphere stimulate extracellular enzyme activity and

increase nitrogen availability in temperate forest soils Biogeochem-

istry 115 65ndash76 httpsdoiorg101007s10533-012-9818-9

Burnham M B Cumming J R Adams M B amp Peterjohn W T

(2017) Soluble soil aluminum alters the relative uptake of mineral

nitrogen forms by six mature temperate broadleaf tree species

possible implications for watershed nitrate retention Oecologia

185 1ndash11

Caporaso J G Kuczynski J Stombaugh J Bittinger K Bushman F

D Costello E K Huttley G A (2010) QIIME allows analysis of

high-throughput community sequencing data Nature Methods 7

335ndash336 httpsdoiorg101038nmethf303

Carreiro M M Sinsabaugh R L Repert D A amp Parkhurst D F

(2000) Microbial enzyme shifts explain litter decay responses to sim-

ulated nitrogen deposition Ecology 81 2359ndash2365 httpsdoiorg

1018900012-9658(2000)081[2359MESELD]20CO2

Cheeke T E Phillips R P Brzostek E R Rosling A Bever J D amp

Fransson P (2017) Dominant mycorrhizal association of trees alters

carbon and nutrient cycling by selecting for microbial groups with

distinct enzyme function New Phytologist 214 432ndash442 httpsdoi

org101111nph14343

Cheng W Parton W J Gonzalez-Meler M A Phillips R Asao S

McNickle G G Jastrow J D (2014) Synthesis and modeling

perspectives of rhizosphere priming New Phytologist 201 31ndash44

httpsdoiorg101111nph12440

Comas L H Callahan H S amp Midford P E (2014) Patterns in root

traits of woody species hosting arbuscular and ectomycorrhizas

Implications for the evolution of belowground strategies Ecology and

Evolution 4 2979ndash2990 httpsdoiorg101002ece31147

Datta R Kelkar A Baraniya D Molaei A Moulick A Meena R S amp

Formanek P (2017) Enzymatic degradation of lignin in soil A

review Sustainability 9 1163 httpsdoiorg103390su9071163

CARRARA ET AL | 11

typically have rapid N cycling rates reflecting greater organic matter

content and root densities than mineral soils (Brzostek amp Finzi

2011) Shifts in the ratio of gross immobilization to gross mineraliza-

tion may also play a role Net N mineralization rates increased in the

OH despite N fertilization induced declines in proteolytic and chiti-

nolytic enzyme activity that produce N monomers for microbial

uptake While we do not have data on gross N cycling we hypothe-

size that declines in gross microbial N immobilization due to reduced

N demand may have outpaced declines in gross N mineralization in

the fertilized watershed Regardless of the exact mechanism it is

important to put the OH results into context on a mass per unit

area basis the OH is less important to total ecosystem N cycling

than the underlying mineral soil

Much of the research on soil N fertilization has focused on

aboveground responses such as litter input and quality or fungal

community shifts to explain widely observed reductions in enzyme

activities and subsequent soil decomposition (Edwards et al 2011

Frey et al 2004 Morrison et al 2016 Sinsabaugh et al 2008

Waldrop et al 2004 Wallenstein et al 2006) Our results provide

evidence that enzyme activity declines under long-term N fertiliza-

tion at the Fernow are driven by a cascade of ecosystem responses

whereby reductions in belowground plant C investment lead to

shifts in bacterial community structure and a decline in their ability

to degrade soil organic matter Thus there is a need to integrate

plantndashmicrobial interactions into our current conceptual and predic-

tive models of N deposition impacts on temperate forests in order

to forecast soil C responses to shifting N deposition regimes

ACKNOWLEDGEMENTS

We acknowledge Mary Beth Adams Tom Schuler and the US Forest

Service staff at the Fernow Experimental Forest for logistical assis-

tance and access to the experimental watersheds and the LTSP site

This work was also supported by the National Science Foundation

Graduate Research Fellowship to Joseph Carrara under Grant No

DGE-1102689 and by the Long-Term Research in Environmental

Biology (LTREB) program at the National Science Foundation (Grant

Nos DEB-0417678 and DEB-1019522) to William Peterjohn Colin

Averill was supported by the NOAA Climate and Global Change

Postdoctoral Fellowship Program administered by Cooperative Pro-

grams for the Advancement of Earth System Science (CPAESS)

University Corporation for Atmospheric Research (UCAR) Boulder

Colorado USA We acknowledge the WVU Genomics Core Facility

Morgantown WV for support provided to help make this publication

possible We also thank Leah Baldinger Brittany Carver Mark Burn-

ham Hannah Hedrick Jennifer Mangano and Catherine Sesa for

assistance in the field and in the laboratory

ORCID

Joseph E Carrara httporcidorg0000-0003-0597-1175

Colin Averill httporcidorg0000-0003-4035-7760

REFERENCES

Adams M B amp Angradi T R (1996) Decomposition and nutrient

dynamics of hardwood leaf litter in the Femow Whole-Watershed

Acidification Experiment Forest Ecology and Management 1127 61ndash

69 httpsdoiorg1010160378-1127(95)03695-4

Adams M B Burger J Zelazny L amp Baumgras J (2004) Descrip-

tion of the fork mountain long-term soil productivity study Site

characterization USDA Forest Service General Technical Report NE

43 323

Adams M B Edwards P J Wood F amp Kochenderfer J N (1993)

Artificial watershed acidification on the Fernow Experimental Forest

USA Journal of Hydrology 150 505ndash519 httpsdoiorg101016

0022-1694(93)90123-Q

Allison S D Wallenstein M D amp Bradford M A (2010) Soil-carbon

response to warming dependent on microbial physiology Nature Geo-

science 3 336ndash340 httpsdoiorg101038ngeo846

Bae K Fahey T J Yanai R D amp Fisk M (2015) Soil nitrogen avail-

ability affects belowground carbon allocation and soil respiration in

northern hardwood forests of new hampshire Ecosystems 18 1179ndash

1191 httpsdoiorg101007s10021-015-9892-7

Brzostek E R Dragoni D Brown Z A amp Phillips R P (2015) Mycor-

rhizal type determines the magnitude and direction of root-induced

changes in decomposition in a temperate forest New Phytologist

206 1274ndash1282 httpsdoiorg101111nph13303

Brzostek E R amp Finzi A C (2011) Substrate supply fine roots and

temperature control proteolytic enzyme activity in temperate forest

soils Ecology 92 892ndash902 httpsdoiorg10189010-18031

Brzostek E R Fisher J B amp Phillips R P (2014) Modeling the carbon

cost of plant nitrogen acquisition Mycorrhizal trade-offs and multi-

path resistance uptake improve predictions of retranslocation Journal

of Geophysical Research Biogeosciences 119 1684ndash1697

Brzostek E R Greco A Drake J E amp Finzi A C (2013) Root carbon

inputs to the rhizosphere stimulate extracellular enzyme activity and

increase nitrogen availability in temperate forest soils Biogeochem-

istry 115 65ndash76 httpsdoiorg101007s10533-012-9818-9

Burnham M B Cumming J R Adams M B amp Peterjohn W T

(2017) Soluble soil aluminum alters the relative uptake of mineral

nitrogen forms by six mature temperate broadleaf tree species

possible implications for watershed nitrate retention Oecologia

185 1ndash11

Caporaso J G Kuczynski J Stombaugh J Bittinger K Bushman F

D Costello E K Huttley G A (2010) QIIME allows analysis of

high-throughput community sequencing data Nature Methods 7

335ndash336 httpsdoiorg101038nmethf303

Carreiro M M Sinsabaugh R L Repert D A amp Parkhurst D F

(2000) Microbial enzyme shifts explain litter decay responses to sim-

ulated nitrogen deposition Ecology 81 2359ndash2365 httpsdoiorg

1018900012-9658(2000)081[2359MESELD]20CO2

Cheeke T E Phillips R P Brzostek E R Rosling A Bever J D amp

Fransson P (2017) Dominant mycorrhizal association of trees alters

carbon and nutrient cycling by selecting for microbial groups with

distinct enzyme function New Phytologist 214 432ndash442 httpsdoi

org101111nph14343

Cheng W Parton W J Gonzalez-Meler M A Phillips R Asao S

McNickle G G Jastrow J D (2014) Synthesis and modeling

perspectives of rhizosphere priming New Phytologist 201 31ndash44

httpsdoiorg101111nph12440

Comas L H Callahan H S amp Midford P E (2014) Patterns in root

traits of woody species hosting arbuscular and ectomycorrhizas

Implications for the evolution of belowground strategies Ecology and

Evolution 4 2979ndash2990 httpsdoiorg101002ece31147

Datta R Kelkar A Baraniya D Molaei A Moulick A Meena R S amp

Formanek P (2017) Enzymatic degradation of lignin in soil A

review Sustainability 9 1163 httpsdoiorg103390su9071163

CARRARA ET AL | 11

De Gonzalo G Colpa D I Habib M H M amp Fraaije M W

(2016) Bacterial enzymes involved in lignin degradation Journal of

Biotechnology 236 110ndash119 httpsdoiorg101016jjbiotec2016

08011

DeForest J L Zak D R Pregitzer K S amp Burton A J (2004) Atmo-

spheric nitrate deposition microbial community composition and

enzyme activity in northern hardwood forests Soil Science Society of

America Journal 68 132ndash138 httpsdoiorg102136sssaj2004

1320

DeSantis T Z Hugenholtz P Larsen N Rojas M Brodie E L Keller

K Andersen G L (2006) Greengenes a Chimera-Checked 16S

rRNA Gene Database and Workbench Compatible with ARB Applied

and Environmental Microbiology 72 5069ndash5072 httpsdoiorg10

1128AEM03006-05

Dix N J amp Webster J (1995) Colonization and decay of wood In N J

Dix (Ed) Fungal ecology (pp 145ndash171) Dordrecht The Netherlands

Springerhttpsdoiorg101007978-94-011-0693-1

Driscoll C T Lawrence G B Bulger A J Butler T J Cronan C S

Eagar C Weathers K C (2001) Acidic deposition in the North-

eastern United States Sources and inputs ecosystem effects and

management strategies AIBS Bulletin 51 180ndash198

Edgar R C (2010) Search and clustering orders of magnitude faster

than BLAST Bioinformatics 26 2460ndash2461 httpsdoiorg101093

bioinformaticsbtq461

Edwards I P Zak D R Kellner H Eisenlord S D amp Pregitzer K S

(2011) Simulated atmospheric N deposition alters fungal community

composition and suppresses ligninolytic gene expression in a North-

ern Hardwood forest PLoS One 6 e20421

Fierer N Lauber C L Ramirez K S Zaneveld J Bradford M A amp

Knight R (2011) Comparative metagenomic phylogenetic and physi-

ological analyses of soil microbial communities across nitrogen gradi-

ents The ISME Journal 6 1007ndash1017

Fierer N Leff J W Adams B J Nielsen U N Bates S T Lauber C

L Caporaso J G (2012) Cross-biome metagenomic analyses of

soil microbial communities and their functional attributes Proceedings

of the National Academy of Sciences of the United States of America

109 21390ndash21395 httpsdoiorg101073pnas1215210110

Finzi A C Abramoff R Z Spiller K S Brzostek E R Darby B A

Kramer M A amp Phillips R P (2015) Rhizosphere processes are

quantitatively important components of terrestrial carbon and nutri-

ent cycles Global Change Biology 21 2082ndash2094 httpsdoiorg10

1111gcb12816

Finzi A C Van Breemen N amp Canham C D (1998) Canopy tree soil

interactions within temperate forests Species effects on soil carbon

and nitrogen Ecological Applications 8 440ndash446

Fog K (1988) The effect of added nitrogen on the rate of decomposi-

tion of organic matter Biological Reviews 63 433ndash462 httpsdoi

org101111j1469-185X1988tb00725x

Freedman Z B Romanowicz K J Upchurch R A amp Zak D R (2015)

Differential responses of total and active soil microbial communities

to long-term experimental N deposition Soil Biology and Biochemistry

90 275ndash282 httpsdoiorg101016jsoilbio201508014

Freedman Z B Upchurch R A Zak D R amp Cline L C (2016)

Anthropogenic N deposition slows decay by favoring bacterial meta-

bolism Insights from metagenomic analyses Frontiers in Microbiology

7 1ndash11

Frey S D Knorr M Parrent J L amp Simpson R T (2004) Chronic

nitrogen enrichment affects the structure and function of the soil

microbial community in temperate hardwood and pine forests Forest

Ecology and Management 196 159ndash171 httpsdoiorg101016jf

oreco200403018

Frey S D Ollinger S Nadelhoffer K Bowden R Brzostek E Burton

A Finzi A (2014) Chronic nitrogen additions suppress decompo-

sition and sequester soil carbon in temperate forests Biogeochemistry

121 305ndash316 httpsdoiorg101007s10533-014-0004-0

Gardes M amp Bruns T D (1993) ITS primers with enhanced specificity

for basidiomycetes - application to the identification of mycorrhizae

and rusts Molecular Ecology 2 113ndash118 httpsdoiorg101111j

1365-294X1993tb00005x

Gilliam F S Turrill N L Aulick S D Evans D K amp Adams M B

(1994) Herbaceous layer and soil response to experimental acidifica-

tion in a central Appalachian hardwood forest Journal of Environmen-

tal Quality 23 835ndash844 httpsdoiorg102134jeq1994

00472425002300040032x

Gilliam F S Welch N T Phillips A H Billmyer J H Peterjohn W T

Fowler Z K Adams M B (2016) Twenty-five-year response of

the herbaceous layer of a temperate hardwood forest to elevated

nitrogen deposition Ecosphere 7 1ndash16

Gilliam F S Yurish B M amp Adams M B (1996) Ecosystem nutrient

responses to chronic nitrogen inputs at Fernow Experimental Forest

West Virginia Canadian Journal of Forest Research 26 196ndash205

httpsdoiorg101139x26-023

Gilliam F S Yurish B M amp Adams M B (2001) Temporal and spatial

variation of nitrogen transformations in nitrogen-saturated soils of a

central Appalachian hardwood forest Canadian Journal of Forest

Research 1785 1768ndash1785 httpsdoiorg101139x01-106

Giovannetti M amp Mosse B (1980) An evaluation of techniques for

measuring vesicular arbuscular mycorrhizal infection in roots New

Phytologist 84 489ndash500 httpsdoiorg101111j1469-81371980

tb04556x

Hassett J E Zak D R Blackwood C B amp Pregitzer K S (2009) Are

basidiomycete laccase gene abundance and composition related to

reduced lignolytic activity under elevated atmospheric NO3- Deposi-

tion in a northern hardwood forest Microbial Ecology 57 728ndash739

httpsdoiorg101007s00248-008-9440-5

Haynes B E amp Gower S T (1995) Belowground carbon allocation in

unfertilized and fertilized red pine plantations in northern Wisconsin

Tree Physiology 15 317ndash325 httpsdoiorg101093treephys155

317

Hernandez D L amp Hobbie S E (2010) The effects of substrate compo-

sition quantity and diversity on microbial activity Plant and Soil

335 397ndash411 httpsdoiorg101007s11104-010-0428-9

Hobbie E A (2006) Carbon allocation to ectomycorrhizal fungi corre-

lates with belowground allocation in culture studies Ecology 87

563ndash569 httpsdoiorg10189005-0755

Hurlbert S H (1984) Psuedoreplication and the design of ecological

experiments Ecological Monographs 54 187ndash211 httpsdoiorg10

23071942661

Jackson C A Couger M B Prabhakaran M Ramachandriya K D

amp Canaan P (2017) Isolation and characterization of Rhizobium

sp strain YS-1r that degrades lignin in plant biomass Journal of

Applied Microbiology 122 940ndash952 httpsdoiorg101111jam

13401

Janssens I A Dieleman W Luyssaert S Subke J A Reichstein M

Ceulemans R Papale D (2010) Reduction of forest soil respira-

tion in response to nitrogen deposition Nature Geoscience 3 315ndash

322 httpsdoiorg101038ngeo844

Johnson N C Gehring C amp Jansa J (2016) Mycorrhizal mediation of

soil Fertility structure and carbon storage Cambridge MA Elsevier

Jones D L Owen A G amp Farrar J F (2002) Simple method to enable

the high resolution determination of total free amino acids in soil

solutions and soil extracts Soil Biology and Biochemistry 34 1893ndash

1902 httpsdoiorg101016S0038-0717(02)00203-1

Kaiser C Kilburn M R Clode P L Fuchslueger L Koranda M Cliff

J B Murphy D V (2015) Exploring the transfer of recent plant

photosynthates to soil microbes Mycorrhizal pathway vs direct root

exudation New Phytologist 205 1537ndash1551 httpsdoiorg10

1111nph13138

Kaiser K Wemheuer B Korolkow V Wemheuer F Nacke H

Scheurooning I Daniel R (2016) Driving forces of soil bacterial

12 | CARRARA ET AL

community structure diversity and function in temperate grasslands

and forests Scientific Reports 6 1ndash12

Klindworth A Pruesse E Schweer T Peplies J Quast C Horn M amp

Gleuroockner F O (2013) Evaluation of general 16S ribosomal RNA

gene PCR primers for classical and next-generation sequencing-based

diversity studies Nucleic Acids Research 41 1ndash11 httpsdoiorg10

1093nargks808

Kochenderfer J N (2006) Fernow and the appalachian hardwood

region In M B Adams D R DeWalle amp J L Horn (Eds) The Fer-

now watershed acidification study (pp 17ndash39) Dordrecht The Nether-

lands Springer

K~oljalg U Nilsson R H Abarenkov K Tedersoo L Taylor A F Bah-

ram M Douglas B (2013) Towards a unified paradigm for

sequence-based identification of fungi Molecular Ecology 22 5271ndash

5277 httpsdoiorg101111mec12481

Lauber C L Hamady M Knight R amp Fierer N (2009) Pyrosequenc-

ing-based assessment of soil pH as a predictor of soil bacterial com-

munity structure at the continental scale Applied and Environmental

Microbiology 75 5111ndash5120 httpsdoiorg101128AEM00335-

09

McCormack M L Adams T S Smithwick E A amp Eissenstat D M

(2017) Variability in root production Phenology 95 2224ndash2235

McGonigle T P Miller M H Evans D G Fairchild G L amp Swan J

A (1990) A new method which gives an objective measure of colo-

nization of roots by vesicular - Arbuscular Mycorrhizal Fungi New

Phytologist 115 495ndash501 httpsdoiorg101111j1469-8137

1990tb00476x

Midgley M G amp Phillips R P (2014) Mycorrhizal associations of domi-

nant trees influence nitrate leaching responses to N deposition Bio-

geochemistry 117 241ndash253 httpsdoiorg101007s10533-013-

9931-4

Morrison E W Frey S D Sadowsky J J van Diepen L T A Tho-

mas W K amp Pringle A (2016) Chronic nitrogen additions funda-

mentally restructure the soil fungal community in a temperate forest

Fungal Ecology 23 48ndash57 httpsdoiorg101016jfuneco201605

011

Oksanen J Blanchet F G Kindt R Legendre P Orsquohara R B Simp-

son G L Wagner H (2015) vegan Community Ecology Pack-

age

Paterson E Gebbing T Abel C Sim A amp Telfer G (2007) Rhizode-

position shapes rhizosphere microbial community structure in organic

soil The New Phytologist 173 600ndash610

Peterjohn W T Adams M B amp Gilliam F S (2017) Symptoms of

nitrogen saturation in two central appalachian hardwood forest

ecosystems Biogeochemistry 35 507ndash522

Phillips R P Brzostek E amp Midgley M G (2013) The mycorrhizal-

associated nutrient economy A new framework for predicting car-

bon-nutrient couplings in temperate forests New Phytologist 199

41ndash51 httpsdoiorg101111nph12221

Phillips R P amp Fahey T J (2005) Patterns of rhizosphere carbon flux

in sugar maple (Acer saccharum) and yellow birch (Betula alleghenien-

sis) saplings Global Change Biology 11 983ndash995 httpsdoiorg10

1111j1365-2486200500959x

R Core Team (2017) R A language and environment for statistical comput-

ing Vienna Austria R Foundation for Statistical Computing

Reay D S Dentener F J Smith P Grace J amp Feely R A (2008)

Global nitrogen deposition and carbon sinks Nature Geoscience 1

430ndash437 httpsdoiorg101038ngeo230

Rousk J amp Baath E (2011) Growth of saprotrophic fungi and bacteria

in soil FEMS Microbiology Ecology 78 17ndash30 httpsdoiorg10

1111j1574-6941201101106x

Saiya-Cork K R Sinsabaugh R L amp Zak D R (2002) The effects of

long term nitrogen deposition on extracellular enzyme activity in an

Acer saccharum forest soil Soil Biology and Biochemistry 34 1309ndash

1315 httpsdoiorg101016S0038-0717(02)00074-3

Schimel J P amp Weintraub M N (2003) The implications of exoenzyme

activity on microbial carbon and nitrogen limitation in soil A theoreti-

cal model Soil Biology and Biochemistry 35 549ndash563 httpsdoiorg

101016S0038-0717(03)00015-4

Sinsabaugh R L (2010) Phenol oxidase peroxidase and organic matter

dynamics of soil Soil Biology and Biochemistry 42 391ndash404 httpsd

oiorg101016jsoilbio200910014

Sinsabaugh R L Antibus R K Linkins A E McClaugherty C A Ray-

burn L Repert D amp Weiland T (1992) Wood decomposition over

a first-order watershed Mass loss as a function of lignocellulase

activity Soil Biology and Biochemistry 24 743ndash749 httpsdoiorg

1010160038-0717(92)90248-V

Sinsabaugh R L Antibus R K Linkins A E McClaugherty C A Ray-

burn L Repert D amp Weiland T (1993) Wood decomposition Nitro-

gen and phosphorus dynamics in relation to extracellular enzyme

activity Ecology 74 1586ndash1593 httpsdoiorg1023071940086

Sinsabaugh R L Lauber C L Weintraub M N Ahmed B Allison S

D Crenshaw C amp Gartner T B (2008) Stoichiometry of soil

enzyme activity at global scale Ecology Letters 11 1252ndash1264

httpsdoiorg101111j1461-0248200801245x

Terrer C Vicca S Hungate B A Phillips R P amp Prentice I C (2016)

Mycorrhizal association as a primary control of the CO2 fertilization

effect Science 353 72ndash74 httpsdoiorg101126scienceaaf4610

Thomas R Q Canham C D Weathers K C amp Goodale C L (2010)

Increased tree carbon storage in response to nitrogen deposition in

the US Nature Geoscience 3 13ndash17 httpsdoiorg101038nge

o721

Toju H Tanabe A S Yamamoto S amp Sato H (2012) High-coverage

ITS primers for the DNA-based identification of ascomycetes and

basidiomycetes in environmental samples PLoS One 7 e40863

Treseder K K (2008) Nitrogen additions and microbial biomass A

meta-analysis of ecosystem studies Ecology Letters 11 1111ndash1120

httpsdoiorg101111j1461-0248200801230x

Vorıskova J Brabcova V Cajthaml T amp Baldrian P (2014) Seasonal

dynamics of fungal communities in a temperate oak forest soil New

Phytologist 201 269ndash278 httpsdoiorg101111nph12481

Waldrop M P Zak D R Sinsabaugh R L Gallo M amp Lauber C

(2004) Nitrogen deposition modifies soil carbon storage through

changes in microbial enzymatic activity Ecological Applications 14

1172ndash1177 httpsdoiorg10189003-5120

Wallenstein M D McNulty S Fernandez I J Boggs J amp Schlesinger

W H (2006) Nitrogen fertilization decreases forest soil fungal and

bacterial biomass in three long-term experiments Forest Ecology and

Management 222 459ndash468 httpsdoiorg101016jforeco2005

11002

Walter C A Adams M B Gilliam F S amp Peterjohn W T (2017)

Non-random species loss in a forest herbaceous layer following nitro-

gen addition Ecology 98 2322ndash2332 httpsdoiorg101002ecy

1928

Wang Q Garrity G M Tiedje J M amp Cole J R (2007) Naive Baye-

sian classifier for rapid assignment of rRNA sequences into the new

bacterial taxonomy Applied and Environmental Microbiology 73

5261ndash5267 httpsdoiorg101128AEM00062-07

Weiss S Xu Z Z Peddada S Amir A Bittinger K Gonzalez A

Hyde E R (2017) Normalization and microbial differential abun-

dance strategies depend upon data characteristics Microbiome 5 27

White T J Bruns T D Lee S B amp Taylor J W (1990) Amplification

and direct sequencing of fungal ribosomal RNA genes for phylogenet-

ics In M A Innis D H Gelfand J J Sninsky amp T J White (Eds)

PCR protocols a guide to methods and applications (pp 315ndash322)

New York NY Academic Press

Yin H Li Y Xiao J Xu Z Cheng X amp Liu Q (2013) Enhanced root

exudation stimulates soil nitrogen transformations in a subalpine

coniferous forest under experimental warming Global Change Biology

19 2158ndash2167 httpsdoiorg101111gcb12161

CARRARA ET AL | 13

Yin H Wheeler E amp Phillips R P (2014) Root-induced changes in

nutrient cycling in forests depend on exudation rates Soil Biology and

Biochemistry 78 213ndash221 httpsdoiorg101016jsoilbio201407

022

Zak D R Holmes W E Burton A J Pregitzer K S amp Talhelm A F

(2008) Simulated atmospheric NO 3 deposition increases organic

matter by slowing decomposition Ecological Applications 18 2016ndash

2027 httpsdoiorg10189007-17431

SUPPORTING INFORMATION

Additional Supporting Information may be found online in the

supporting information tab for this article

How to cite this article Carrara JE Walter CA Hawkins JS

Peterjohn WT Averill C Brzostek ER Interactions among

plants bacteria and fungi reduce extracellular enzyme

activities under long-term N fertilization Glob Change Biol

2018001ndash14 httpsdoiorg101111gcb14081

14 | CARRARA ET AL